Sex determination genes and their use in breeding

ABSTRACT

The invention relates to a method to improve breeding in dioecious plants, preferably  Asparagus  plants, comprising providing a plant in which the functional expression of the dominant suppressor of gynoecium development is disrupted or reduced and introducing said plant in inbreeding, backcross breeding, recurrent backcross breeding or double haploid breeding techniques. Preferably said dominant suppressor of gynoecium development is a gene comprising a DUF247 domain. Also provided are dioeciuos plants in which the expression of this gene is disrupted or reduced.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the national phase of PCT applicationPCT/IB2016/000031 having an international filing date of 10 Jan. 2016,which claims benefit of Dutch patent application No. 2014107 filed 9Jan. 2015. The contents of the above patent applications areincorporated by reference herein in their entirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 313632021800SeqList.txt,date recorded: Jun. 2, 2020, size: 945,059 bytes).

FIELD OF THE INVENTION

The present invention relates to the field of plant breeding, inparticular breeding of dioecious plants, in particular asparagus. Theinvention extents to the field of both classical and molecular plantgenetics and relates to sequences of a novel DUF247 motif containinggene and its mutants and their use in marker assisted breeding, targetedmutagenesis, or in transgenic plants, e.g. to produce feminized orde-feminized plants. It further relates to sequences of the asparagusgene homologous to the Arabidopsis TDF1 gene (AT3G28470) or the Oryzasativa osTDF gene (LOC_Os03g18480), and their use in marker assistedbreeding, or in transgenic plants, e.g. to produce masculinized orde-masculinized plants.

BACKGROUND OF THE INVENTION

Plant breeding is one of the oldest accomplishments of mankind. It beganwhen he domesticated plants by growing them under controlled conditionsand selecting those types that provided a dependable source of food. Noproduct of the plant breeder's art or science has had greater impact onincreasing the world's feed or food resources than hybrid varieties.Dramatically successful at first in corn, their use has spread to othercrops, including both cross- and self-pollinated species. Hybridvarieties are those in which F1 populations are used as the commercialcrop. Parents of the F1 may be inbred lines, clonal varieties or otherpopulations. Hybrid varieties are used where the increased yield fromhybrid will be more than from the extra costs associated with theirdevelopment and the extra costs of their seed production price. An addedpremium in the case of hybrids of inbred lines is uniformity. Methodsfor developing hybrid varieties are provided in the book “Introductionto Plant Breeding” by FN Briggs and PF Knowles (1967) (supra p 223.239).

Plant breeding has the objective to produce improved crop varietiesbased on the exploitation of genetic variation, which exists within thegermplasm of a plant species. Genetic variation is traditionallyobtained by crossing two genetically distinct plants to create hybridprogeny. In the process of developing hybrid varieties, hybridization isnot aimed at producing a pure-breeding population but rather to produceF1 hybrid plants as the final cultivar.

The F1 hybrid of crosses between different genotypes is often much morevigorous than its parents. This hybrid vigour, or heterosis, can bemanifested in many ways, including increased rate of growth, greateruniformity, earlier flowering, and increased yield, the last being ofgreatest importance in agriculture

The production of hybrid varieties commonly involves three steps: (1)the selection of superior plants; (2) inbreeding for several generationsto produce a series of inbred lines, which although different from eachother are each pure-breeding and highly uniform; and (3) crossingselected inbred lines. During the inbreeding process the vigour of thelines decreases drastically compared to that of field-pollinatedvarieties. Vigour is restored, however, when any two unrelated inbredlines are crossed, and in some cases the F1 hybrids between inbred linesare superior to open-pollinated varieties. An important consequence ofthe homozygosity of the inbred lines is that the hybrid between any twoinbreds will always be the same. Once the inbreds that give the besthybrids have been identified, any desired amount of hybrid seed can beproduced.

As outlined above, an essential step in creating a hybrid cultivar is toobtain inbred lines. In non-dioecious crops, a common way to obtainthese homozygous plants is to apply self-pollination andself-fertilization for several generations (inbreeding). Alternatively,the process of inbreeding by several generations of self-fertilizationcan be replaced by creating plants exclusively derived from gametes,either egg cells (gynogenesis) or from pollen (androgenesis). When thegenetic content of plants derived from gametes is doubled, either bychemical means (such as by using colchicine) or by spontaneouschromosome doubling, fully homozygous plants are obtained. Such plantsare called doubled haploids. In non-dioecious crops such as pepper,eggplant, cucumber, maize, rapeseed, broccoli etc. —doubled haploids canbe multiplied by seed propagation, simply by self-fertilizing suchplants. This allows fast multiplication of parental lines which ishighly desirable when used as parental plants in large scale hybrid seedproduction. Another advantage of seed propagation of doubled haploids isthat it allows convenient storage, as seeds can be stored for arelatively long time under controlled climate conditions in relativelysmall compartments. Compared to storage of living plants, that requireland or greenhouse space, and that are prone to adverse environmentalconditions, pathogen attack, and somatic mutations, seed storage isrelatively safe at low costs. Furthermore, seed propagation can be usedto get rid of certain (non-seed transmittable) pathogens. In addition,seed propagation may improve plant growth of ex-vitro plants which maygrow sub-optimal as a result of the long lasting effect of hormonesapplied during tissue culture (Smulders & de Klerk, 2011) in a way itcould restore the lowered DNA methylation that resulted from tissueculture (Machczynska et al., 2014) although some methylation changes maybe heritable (e.g. see Stelplug et al, 2014). In this sense, seedpropagation could positively change the physiological state of explants.Clearly, the ability to reproduce doubled haploids by seed propagationoffers several advantages. In the production of doubled haploids in thedioecious crop Asparagus, anther culture (Qiao & Falavigna 1990) ormicrospore culture (Peng & Wolyn, 1999) is applied and there are noreports of successful in vitro gynogenesis in Asparagus. As aconsequence of this, in vitro haploid production is restricted to maleplants, thus those plants that are capable of producing functionalanthers. The inability to self-fertilize and/or to apply in vitroandrogenesis hampers the improvement of seed parents of commercialhybrids in case those seeds parents showed a good combining ability inearly generation hybrid testing (for early testing see: Longin et al.,2007). This contrasts to the situation in non-dioecious crops such ascorn, pepper, eggplant, brassica etc. where a seed parent of a hybridcultivar can be directly improved, either by further inbreeding orhaploid production using a single plant as starting point. Inconclusion, inbreeding and/or seed multiplication by self-pollinationand in nitro androgenesis is fully obstructed for female asparagusplants. Direct in vitro androgenesis cannot be applied to female plantssuch as the seed parents of asparagus hybrids.

Besides inbreeding or doubled haploid production as a tool to createelite hybrid parental lines, breeders can use other techniques. One suchtechnique is referred to as back-cross breeding or recurrentback-crossing. In back-crossing a donor parent, which has one or moregenes of interest, is crossed to a recurrent parent which is an eliteline that could be improved by adding such one or more genes ofinterest. The progeny of this cross is selected for the trait ofinterest and then crossed back to the recurrent parent. This process isrepeated for as many back crosses as are needed to create a line that isgenetically similar (syngeneic) to the recurrent parent, except—ofcourse—for the gene(s) of interest. The goal of backcrossing is toobtain a line as identical as possible to the recurrent parent with theaddition of the gene(s) of interest that has been added through thisbreeding process. Recurrent back-crossing or back cross breeding is anefficient way to improve the quality of parental lines that are known tocombine well as parents of hybrids but hitherto lacked certain traits tomake these even more perfect parental lines. In non-dioecious crops itis irrelevant whether a trait needs to be introduced in a breeding linethat will finally serve as the female parent or the male parent of ahybrid. However, in a dioecious crop, such as asparagus, a first crossto introduce a trait found in a female donor plant into the seed parentof a hybrid is impossible as females cannot be crossed with females.Likewise, most male plants (andromonoecious plants excepted) cannot beintercrossed in a dioecious crop, such asparagus, therefore a firstcross to start a backcross program to introduce a trait from a maleplant into another male is not, possible. Further below it, will beexplained that backcrossing to introduce a trait in the male parent ofan all-male hybrid is problematic, even in the case that the donor ofthe trait is a female plant, for a dioecious crop such as asparagus.

Generally, the breeding tools outlined above, such as the [1] ability toapply self-fertilization, [2] the ability to apply successivebackcrossing, [3] the ability to apply seed propagation and/or seedstorage of doubled haploids or inbred lines or [4] the ability tofurther improve the seed parents of an early generation hybrid by invitro androgenesis can be used in many non-dioecious crop species (e.g.corn, pepper, rapeseed, cabbage, cauliflower, broccoli sunflower,barley, cucumber, eggplant). However, the dioecious, rather thanhermaphrodite nature of Asparagus officinalis, limits the use ofself-fertilization, back-crossing, and seed propagation of doubledhaploids and in vitro androgenesis of hybrid seed parents in asparagusbreeding. There is thus need to provide methods to, at least partly,overcome the limitations caused by dioecy on breeding and seedproduction of asparagus. To appreciate this, one should he aware of allaspects on the inheritance of gender in Asparagus officinalis and theuse of so called ‘super males’ to create all-male asparagus hybrids.This will be further explained below. Before describing the inheritanceof gender traits first some definitions are made that will allow thereader to better understand the text below. A female asparagus plant isa plant that produces only flowers that have fully developed femaleorgans, such as a style and stigma that allows fruit set and onlyproduces white rudimentary anthers. A male asparagus plant is capable ofproducing flowers with fully developed anthers. If a male plant iscapable of producing berries it is either andromonoecious orhermaphrodite. Andromonoecious plants bear both male flowers that onlyhave rudimentary female organs and hermaphrodite ‘perfect’ flowers,whereas hermaphrodite plants exclusively produce hermaphrodite flowers.One would expect that a highly andromonoecious plant but at least a truehermaphrodite plant will produce berries from virtually every flower.However, as will be further discussed below, this is not always true forplants typed as hermaphrodite (Thevenin, 1967) or not recorded forhighly andromonoecious plants (Wricke, 1968, Wricke, 1973) which israther confusing.

Asparagus officinalis is a dioecious species with separate unisexualindividuals producing male or female flowers. Male and female flowers atearly stages of development possess both carpels and stamens; sexdifferentiation appears to be the result of the selective abortion ofcarpels in male flowers and of stamens in female flowers. The abortionpattern is, however, different in the two sexes: in female flowers,stamens stop developing and collapse while, in male flowers, the ovaryremains blocked in its growth without degenerating after stamens havetaken over (Lazarte and Palsen. 1979, Caporali et al., 1994).

The genetic control of sex determination in this plant is based on amodel in which regulatory genes control the expression of structuralgenes involved in stamen and carpel development present in both sexes.Two regulatory genes of the type “male activator” and “femalesuppressor” as proposed by Westergaard (1958) in Silene, have beensuggested to be operating in A. officinalis. One of the first authorswho raised the model of Westergaard as a model for sex determination inasparagus was Wricke (1968). In the introduction of his publication theauthor describes female and male asparagus plants that are homogamous XXand heterogamous XY, respectively and further notes the possibility toobtain homogamous YY male plants by self-fertilization (also proposed byRick & Hanna 1948, and Sneep 1953). This self-fertilization is possibleas a small fraction of male plants is able to produce hermaphroditeflowers. YY male plants, also referred to as ‘super-males’ allow theproduction of entirely female free cultivars, also referred to as‘all-male hybrids’, when those plants are crossed to female plants. Anall-male cultivar is particularly valuable if plants belonging to thiscultivar produce no berries at all and this poses a conflict. When theability to produce YY males by self-fertilization of a plant thatproduces hermaphrodite flowers is heritable, it is likely that thistrait will be transferred to the hybrid, which is undesirable. Thequestion to which extent the relative amount to produce hermaphroditeflowers is heritable was raised earlier by Beeskow (cited in Wricke,1968) who classified flowers, that all have anthers, into types denotedby the roman numbers I, II, III, and IV to describe the stages of aflower that has no style or stigma at all (I) up to a flower that has afully developed style and stigma: (IV). Wricke (1968) explains that thematerial he studied could be divided into two groups, in which one grouppredominantly produces flowers of type IV (and never flowers of type 1),whereas the other group predominantly produces flowers of type I (andnever flowers of type IV). Based on the fact that some males crossed toa particular female result in progeny that predominantly produce type IVflowers whereas other males crossed to the same females result inprogeny that predominantly produce type I flower (see his Table 1)Wricke, (1968) concluded that a major factor on the Y chromosome confersthe ‘andromonoecy-degree’. This interpretation is subject to debate.Although his data indeed show that the level of andromonoecy seems todepend on the particular paternal plants chosen, these results do notreject that this may result from mere chance (thus not necessarilydepends on the parental, rather than any parent) as only a limited setof female plants has been used (six maternal plants versus twentydifferent paternal plants).

Wricke tackles the controversial interpretation of the results shown inhis Table 1 (published in 1968) by presenting a new Table 1 in afollowing publication (Wricke, 1973) in which he shows the result ofsecond generation pedigrees of crosses between females and males thateither were members of pedigrees showing high or low levels ofandromonoecy in their previous pedigree. Indeed results obtained fromthose pedigrees presented in Wricke (1973) suggest that factorsconferring andromonoecy must reside on the Y chromosome. In hispublication of 1968, Wricke, regularly (as also shown in the subtitle ofhis paper ‘Ein Majorfaktor für die Ausprägung des Adromonoöziegrades’)mentions a major factor on the Y chromosome which confers andromonoecyand he seeks evidence for this hypothesis by a detailed discussion ofresults obtained from the highly andromonoecious plant ‘143/4a/5’. Thisplant (that itself shows flowers of type III and type IV) was crossed tothree mother plants. In those crosses, andromonoecious plants (type IV)and female plants were obtained. When plants, within the progeny thatdid not flower were interpreted as females (which usually show delayedflowering) this corresponded to a 1:1 ratio for plants that segregatefor the absence or presence of anthers but breed true for awell-developed style and stigma. Plant 143/4a/5 was further crossed tothree father plants that were all hypothesized to have a low level ofandromonoecy. The resulting progenies (designated 4, 5, and 6) lackedfemales and comprised both male (class I) and andromonoecious plants(class VI). Wricke (1968) interprets these result as the gene action inwhich a ‘Y_(I) chromosome dominates over the Y_(IV) chromosome’consistent with a dominant female suppressor, proposed for Silene(formerly Melandrium) by Westergaard (1958) who is cited in his paper.However. Wricke's (1968) results obtained for progenies 5 and 6,hypothesized to be XY_(IV)×Y_(I)Y_(I), in fact are inconsistent withthis model; plenty of andromonoecious plants were observed which,theoretically cannot exist because of the assumed dominance of thehomozygous father (Y_(I)Y_(I)). The self-fertility of Wricke's materialremains obscure as he does not describe the level of berry set andsubsequent seed production in both of his publications. In Wricke.(1973) it is even explicitly mentioned that fruit set has not beenrecorded. Another limitation of the work of Wricke (1973) is that heonly describes the average level of andromonoecy in his Table 1 and doesnot supply the segregation ratios for andromonoecy within thosepedigrees. In conclusion, the work of Wricke (1968, 1973) providesinsufficient teaching on the exact mode of inheritance of andromonoecyand the presented data does not (fully) support his conclusions.

A second study in which the model of Westergaard (1958) is raised as amodel for sex determination in Asparagus is the work of Thevenin (1967).This author describes three flower types. Type 1 represents a femaleflower with a well-developed pistil, a tri-lobular stigma and whiterudimentary anthers. Type 2 represents hermaphrodites that have a pistilcomparable to that of female flowers and six yellow stamens at allpoints comparable with those of males. Type 3 represents flowers of amale type or flowers that have an intermediate phenotype and have a moreor less reduced pistil (an ovary of reduced size, a reduced style oreven no style (zero), stigmas that have one or two lobes and a reducednumber of papilla or none). She further states that plants that hearfemale flowers hear no other types, which also holds for plant bearingflowers of Type 2. In conclusion, Thevenin (1967), describes plants forwhich every flower can be perfect. However, it is noted that theproduction of berries and seeds does not depend fully on flowermorphology except for flowers of Type 1. In the work of Thevenin it isexplained that usually, plants that only bear Type 2 flowers produce anumber of berries that may vary from zero to one up to several thousandsalthough the latter number is exceptional. It is important to note thatit remains obscure from the work of Thevenin (1967) whether she everfound a hermaphrodite (as inferred from the flowering) that sets fruitfrom each flower. The hermaphrodites described produce small berriesthat usually contain only single seeds. Thevenin (1967) points out thathermaphrodites usually produce at least some seeds that have animperfect (white) seed-skin as opposed to black perfect seed skin thatis commonly found in female plants By allowing uncontrolled as well ascontrolled self-fertilization, Thevenin (1967) obtained progenies forplants that were either classified as Type 2 or Type 3 (based on theflowers observed on those plants). Each of those progenies segregatedfor female, male and bi-sexual plants. The fact that both male andbisexual plants are found in this progeny poses a problem. Like Wricke(1968), also Thevenin adopts the model of Westergaard (1958). Shehypothesized that these bi-sexual plants result from a crossing-overevent that resulted in a pair of linked genes [M su] where ‘M’ denotes agene involved in anther development, and ‘su’ a recessive allele of adominant female suppressor Su that is commonly linked to M. If accordingto this model [M su/M su] plants are self-fertilized, theory excludesthe presence of [M su/M su] males in the progeny, which however havebeen found in the study of Thevenin (1967). To explain this, Theveninintroduces a series of recessive genes ‘r’ that in homozygous conditionnegatively interfere with stigma development in plants that carry thedominant M allele, thus only in male plants.

The last person who describes the genetic mechanism for sexdetermination in asparagus as the result of two linked genes is Marks(1973). Although this author does not explicitly refer to Westergaard(1958) an equivalent model is presented in which a recessive gene ‘g’controls the gynoecium and is closely linked with a dominant gene ‘A’concerned with the androecium. Marks (1973) states that this model ismore appropriate compared to other models ‘as it requires nomodification or very little to explain the results obtained withhermaphrodites and to state his case, he uses the data that werepreviously obtained by Peirce and Currence (1962). These latter authorsdescribe hermaphrodite cultivated asparagus plants, which have perfectflowers and performed crossing experiments to unravel the inheritance ofhermaphroditism. It was the conclusion of Peirce and Currence (1962)that hermaphroditism is controlled by several dominant linked geneslocated on the sex chromosome separated by a crossing over distance of30 to 40 cM. Thus compared to the interpretation of Peirce and Currence(1962) who report of several dominant genes, the model of Marks (1973)who reports of a dominant gene for the androecium development combinedwith a linked recessive gene for gynoecium development is quitedifferent. Although Marks’ (1973) model fits for two pedigrees (‘2-3’and ‘3-4’) his model fails to explain the existence of males in twoother pedigrees (‘1-6’ and ‘4-1’). These males are then explained byMarks (1973) ‘as being genetically hermaphrodite as far as the gA locusis concerned but having in addition a recessive gene s which whenhomozygous suppresses the gynoecium; such plants then beingphenotypically male’. In this sense, his model is polygenic just likethe model of Thevenin (1967). The inconsistencies between thetheoretical ratios and the observations in the second generation F₂ andBC₁ pedigrees stemming from pedigree ‘2-1’ are explained in Marks (1973)as the result of distorted segregation. Although he claims to provide amodel that in his view ‘requires no modification or very little toexplain the results obtained with hermaphrodites’ it is—like othermodels—still based on explanatory hypothesis such as a recessivemodifier and distorted segregation that have not been tested further.Marks (1973) replies that more data would needed to a question ofThevenin in a discussion section guiding his paper (page 129) on how toverify his hypothesis. Personal communication with University of NewHampshire emeritus Professor Lincoln C. Peirce (e-mails 2010 and 2015)indicates that more unpublished data have been obtained, which indicatethat the experimental evidence obtained for pedigree 2.3, notablyfurther generations, have been less clear than would arise from themodel suggested by Marks (1973). Lincoln C. Peirce has stated thefollowing: ‘After the original work was done and published, I continuedto make crosses, hoping to learn more about the inheritance system. Themore crosses or self-pollinations I made, the more inconsistencies Ifound, all from material derived from the original crosses orbackcrosses’. And he further wrote: ‘‘2.3’ was unique—I never found aplant like it in later crosses or selfs. That led me to conclude thatthere had to be other factors involved, but I never was able to pursueit.’

In a question to elaborate on the uniqueness of 2-3 Lincoln C. Peircereplied that he ‘never found any lines just like 2.3’ in a sense that‘there never was found any derived line that was as stronglyhermaphroditic as 2-3 where every flower produced a berry yet had fullydeveloped anthers’.

These unpublished results contrast with the model of Marks (1973)which—based on a recessive gene ‘g’ that controls the gynoecium that isclosely linked with a dominant gene ‘A’ concerned with theandroecium-predicts that plants as strong as 2.3 would be observed inlater progenies.

In conclusion, in each study in which the model of Westergaard (1958),that describes a dominant female suppressor, is supposed to be acting inAsparagus (Lopez-Anido and Cointry 2008) the results do not fully complywith this model or to put it more strictly, the results would rejectthis model.

Earlier work before the model of Westergaard was published, originatesfrom Sneep (Sneep, 1953a, 1953b). This author, who strongly advocatedthe use of andromonoecious plants to obtain super males as hybridparents, acknowledged the importance to prevent andromonoecy incommercial seed and performed genetic analysis of the trait to possiblytackle this problem. One andromonoecious sibling in the progeny hedescribes, which spans three generations, breeds true for this traitwhereas another andromonoecious sibling was able to produce purely maleindividuals, besides andromonoecious individuals. As a result Sneep(1958b) concludes that andromonoecy is controlled by dominant factorsand that progeny size has been too small to predict the number offactors involved. As method to prevent andromonoecy he suggest to selectplants that have recessive alleles for dominant genes controllingandromonoecy.

Another model that resembles, yet only partly overlaps, with the modelof Westergaard (1958) is a model proposed by Franken (1970). This authorstudied several progenies of self-fertilized andromonoecious plants andconcluded that a partial dominant gene (modifier, ‘A’ in hisnomenclature) was responsible for the suppression of pistil development(see Table below); and that this gene was inherited independently of themale sterility allele located on the X chromosome.

Phenotypes (genders) and genotypes proposed by Franken (1970) as a modelof inheritance of sex.

Female Male Andromonoecious XX AA XY AA XY Aa; weakly XX Aa YY AA XY aa;strongly XX aa YY Aa YY aa; medium

The model proposed by Franken (1970 is in concordance with the resultsof Galli et al. (1993) who, after analyzing the length of pistils insome backcrosses, concluded that the factors affecting style length andstigma development (modifiers) are not localized on the sex chromosome.In the model of Galli et al (1993) the backcross distribution of stylelength fitted a model of at least two loci. Franken's model, presentedin the table above principally is an additive genetic model ofcomplementary gene action in which the Y chromosome gives rise tostaminate flowers (anthers but no pistils) and recessive ‘a’ allelesalleviate the effect of the Y chromosome and allow some pistildevelopment. The balance between the number of Y chromosomes that pushflowers in the staminate direction (anthers but no pistil) and thenumber of recessive ‘a’ alleles, which allow a certain degree of pistildevelopment, sets the level of perfect flowers that can be produced.Franken (1970) acknowledged that not all of his crossing results couldbe explained by a simple genetic model and careful study of thetabulated results in Franken's PhD thesis (see Table 37a and Table 37bin Franken, 1969, chapter 8 pp. 56-58) suggests that andromonoecy is aquantitative rather than a qualitative trait which can be influenced bythe environment. To meet this quantitative aspect, especially to explainYYAa plants that sometimes tend to become more andromonoecious, Franken(1969, 1970) introduced G factors that positively contribute to stigmadevelopment in males. Thus like Sneep (1953b), also Franken (1969, 1970)describes dominant genes that may contribute to andromonoecy.

All of the above studies have demonstrated that principally, a maleplant can be produced by self-fertilization of flowers onandromonoecious plants, which plants sometimes were referred to ashermaphrodite when all flowers were perfect. It was further explainedthat if a andromonoecious plant (XY) is self-fertilized, a quarter ofthe progeny will be YY; in asparagus breeding referred to as a supermale. In case a super male is used as paternal parent to pollinate afemale parent, hybrid progeny is obtained of which all plants are XY,thus are male in a way that all of these plants will produce anthers.However, whether or not those plants will be able to produce berrieswill rely on multiple factors such as ‘Su/su’ and ‘r’ (Thevenin, 1967),‘Y’, ‘Ala’, and ‘G’ (Franken, 1969, 1970), ‘several dominant factors’(Sneep, 1953a, 1953b, Peirce & Currence, 1961) and ‘Su^(F)/su^(F)‘plusmodifying genes that either moderately or strongly control stigmadevelopment’ (Wricke, 1967. p209) and ‘a recessive gene s which whenhomozygous suppresses the gynoecium’ or a phenotype frequency that maybe influenced by distorted segregation (Marks, 1973).

All these modifying genes or factors, of which some may designate thesame gene or factor, are unknown. As already pointed out by Sneep, theandromonoecious trait used to create super-males must not end up incommercial seed. This poses a conflict: the parental lines of a hybridcultivar can be created by self-fertilization mediated by heritableandromonoecy and the more this heritable trait is expressed, the moreefficient the creation of lines will be. However, the hybrid thatoriginates from a cross between such parental lines should not expressthe heritable trait. If the heritable trait is complex and unknown, thebreeder is unable to exploit expression to create inbred parental linesof a hybrid on the one hand, and avoid expression on the other hand,when the parental lines are used to create a commercial hybrid.

As a result breeders preferably avoid obtaining YY plants by selfingandromonoecious plants but instead prefer to obtain these by antherculture of male plants. However, even in the case of using doubledhaploids as parental plants, hybrids can be created that areandromonoecious when parental plants either from the maternal side orpaternal side have piled up a sufficient number of modifiers thatovercome the hypothesized masculinizing effect of the Y chromosome (ashypothesized by Franken, 1970). It should further be noted that if thebreeder would like to apply inbreeding by using andromonoecious plantsthis work is limited by the fact that andromonoecy is restricted to asmall subset of the genepool or germplasm as andromonoecy orhermaphroditism occurs in about 0.1 up to 2% of the breeding material(Thevenin, 1967, Sneep, 1958). In conclusion the breeder must avoid thatmodifiers end up in the hybrids and further the breeder is limited bythe rare availability of sufficient andromonoecy throughout the breedingpool.

Whether or not super males have been obtained by self-fertilizationusing andromonoecy or anther culture, the super males created havecertain shortcomings compared to male parents of hybrids of cropsbelonging to common self-pollinating species (such as tomato, pepper,eggplant, rapeseed, broccoli etc). Firstly, because genes that favorablymodify the phenotype towards andromonoecy must be avoided (else hybridswould produce unwanted berries) which means that super males can neverbe seed propagated at large scale.

Secondly, and this is an important aspect, a super male cannot beimproved by successive backcrossings as the F1 plant obtained in thefirst cross is a male that cannot be directly backcrossed to the supermale in which a new trait should be introduced.

In case a breeder would like to make use of perfect flowers that allowself-fertilization, at least simple inheritance of the hermaphroditetrait would be desirable, preferably a monogenically inherited trait,which is easy to get rid of in one or just a few generations.Preferably, such a monogenic trait can be selected for by a geneticmarker.

In conclusion, the art of asparagus breeding would strongly benefit fromthe availability of hermaphroditism that is simply inherited and thus ishighly predictable and easily selected for or selected against incertain stages of breeding, preferably by a genetic marker.

Further the art of asparagus breeding would strongly benefit if abreeder could use a method that allows inbreeding by self-fertilization,and allows to seed-propagate inbred lines or to seed propagate doubledhaploids. Finally, an asparagus breeder would like to be able to applydirect recurrent backcrossing on a super male plant as recurrent parent.A breeder would like to be able to perform all of the above withoutbeing bothered by introducing unknown modifiers, unlinked to the sexchromosome, that favor andromonoecy or being bothered by the limitednumber of plants that exhibit sufficient natural andromonoecy. Ideally,the change from a male plant into a hermaphrodite, or, more generally,methods to influence the sex of plants in a breeding scheme, to tackleall of the above problems, is targeted and acts temporarily.

In an even more ideal situation the female suppressor or suppressor ofgynoecium development, that is hypothesized but never fully proven toexist or at least not proven to act monogenically is identified and canbe manipulated in a sense of ‘switching it on and off’.

Where a breeder could be interested in enabling or disabling gynoeciumdevelopment this breeder—depending on the intended use of a plant aseither seed or pollen parent or both—could also be interested inenabling androecium development. Enabling androecium development in afemale plant to essentially change the gender, would allow to obtainseeds from an originally female plant in the absence of crosspollination (thus by self-fertilizaton) and would provide the ability toobtain doubled haploids by in vitro androgenesis from such a plant. Thiswill allow inbreeding which may led to breeding lines that are moresuperior compared to the original female plant that was enabled toself-pollinate. It will allow seed storage of the female breeding line.The ability to tune and change the gender of female plants (originallylacking functional anthers) and male plants (originally fully or partlylacking gynoecium development) will allow flexibility in crossingschemes that are currently hampered by dioecy. The ability to tune andchange the gender of male and female plants may also broaden the genepool in creating hybrids when a male plant that appears to be a goodgeneral combiner in hybrid crosses could be changed into a female plantand then crossed to suitable male plants or when a female plant thatappears to be a good general combiner in hybrid crosses it can bechanged into a male plant and then can be crossed to female plants.

In the art, its has been suggested that such sex changes might occur(Maeda et al 2005) but the evidence thus far has been too weak to beabsolutely sure this has happened, let alone to understand how this canbe accomplished.

In a study published by Maeda et al (2005) it has been hypothesized thata female asparagus plant has been obtained from the male vitroclonecultivar Festo as the result of in vitro embryogenesis. It is writtenthat a sex conversion has been ‘identified’ and that ‘the sex conversionin the current study might be the result of the somatic mutation, suchas somatic crossing-over, one of the chromosomal rearrangements’. Thishypothesis of sex conversion is based on the genetic analysis of asingle female plant found in an evaluation field that was compared tofive male plants. Maeda et al (2005) have used allozymes that werepreviously used by Ozaki et al (2000a), where Ozaki has been thecorresponding author for both studies. Ozaki et al (2000a) discussed theuse of allozymes repeatedly in the light of detecting contamination.However, rather than testing plant contamination, Maeda et al (2005)discussed their results only in the light of multiple somaticrecombination events and have projected the loss of heterozygosity,notably the change of the Mdh1 allozyme locus loosely linked to the Mlocus, on a theory of a sex conversion. It appeared that essentially forhalf of the loci tested the observed female genotype was differentcompared to the males it was expected to be derived from. The authorshave tested eight allozyme loci where they found that a female plant wassimilar to the male plant at five loci: “bb” in Aat I “aa” in Aat 2 “bb”in Aat-3 “bc” in Pgm-1 and “ab” in Skdh-1. This is a low number of lociand it seems that the discriminative power of two of these similar loci,Aat I and Aat 2 may have been limited. Ozaki et al. (2000a) have shownthat for nine cultivars tested, only two alleles were observed for Aat Iand that the ‘bb’ homozygous genotype was observed in eight of these. Itfurther seems that Aat 2 showed no variation at all. It should furtherbe noted that two other loci Pdm-1 and Skdh-1 are closely linked (4-6cMsee Ozaki et al., 2000a and reference therein) which also limits thediscriminate power as these loci markers essentially target a similarlocus. At another three loci differences were observed for the malecontrols plants vs the female plant, respectively; “an” vs. “nn” in MdhI “an” vs. “un” in Mdh 2 and “an” vs. “aa” in Idh I.

The authors reasoned that the sex-conversion might be the result of thechange of the genotypes from “Mm” to “mm” in sex determining locus andthat ‘Mdh-1 and Idh-1 changed to be homozygous from heterozygous inaccordance with the mutation’, Besides that this is highly speculative,the theory seems to be based on assumptions that are factually wrong.The authors cite the work of Maestri et al. (1991) which demonstratedthe linkage of MdhI to the M-locus and this is indeed disclosed in thecited work. The authors also state that ‘three linkage pairs ofAat-1/Mdh-1, Aat-1/Idh-1 and Pgm-1/Skdh-1 were recognized previously(Ozaki et al, 2000b)’, but this is incorrect as Ozaki et al (2000b)found Idh1 to be linked to Aat3 rather than Aat1.

Therefore the scenario that both loci ‘changed in accordance with themutation’ as if this would be an obvious event effecting (a part of) oneand the same chromosome is not supported. To gain at least some evidencethat Mdh1 loss of heterozygosity is connected to a mutation that couldbe associated with a hypothesized sex conversion, it must be establishedthat the allegedly lost allele observed in the female has been linked incoupling phase or ‘in cis’ to the dominant M allele conferring the malephenotype in the original cultivar. Testing such an hypothesis is easilyperformed by a testcross such as made by Maestri et al. (1991) using afemale that preferably homozygously differs at the MdhI locus from thecultivar Festo. Such an experiment has not been performed by Maeda etal. (2005) and this leaves the connection, in the sense of a beingcausal, between an allegedly lost MdhI allele and the alleged sexconversion unsolved.

The third variable locus, Mdh2, has not been found to be linked to Mdh1(see Ozaki et al., 2000b).

An proprietary marker of Limgroup targeting an Mdh gene:

(SEQ ID NO: 11) CAGCTATAGGGACGGTAGAATTTAC[C/T]GGGTTGCTAATGATGTGAAT GAwas found to be linked to Asp276:

(SEQ ID NO: 12) GTAGATTCAAGGGAGTACGGCATTGGCGCGCAGATATTGCACGATCTTGG[C/T]GTTCGGACAATGAAGTTGCTGACCAACAACCCGGCAAAATATAGC GGGCTthat was mapped to a chromosome designated chromosome 8 in a proprietymapping population, rather than to the sex chromosome.

This reconfirmed that Mdh1 found to be linked to the M locus and Mdh2are not linkedMaeda et al. (2005) could have provided more conclusivedata with this respect e.g. to clarify whether contamination that couldhave been inferred from the observed genetic variability has been afrequent event in the evaluation field. It is not explained why thissecond plants has not been tested.

In conclusion, the report of a sex conversion that according to Maeda etal (2005) has been identified, will be subject to debate to the personskilled in the art and will raise many unanswered questions to theperson skilled in the art and thus provides insufficient teaching onwhether a sex converted asparagus plant can he obtained by in vitroembryogenesis.

Hence, the breeder of monoecious plants, especially Asparagus plants isstill in need of the availability of hermaphroditism that is simplyinherited and thus is highly predictable and easily selected for orselected against in certain stages of breeding. Further, the skilledbreeder would also be interested in enabling androecium development.Enabling androecium development in a female plant to essentially changethe gender, would allow to obtain seeds from an originally female plantin the absence of cross pollination (thus by self-fertilizaton) andwould provide the ability to obtain doubled haploids by in vitroandrogenesis from such a plant.

SUMMARY OF THE INVENTION

The present invention relates to a method to improve breeding indioecious plants comprising providing a plant in which the functionalexpression of the dominant suppressor of gynoecium development isdisrupted or reduced and introducing said plant in inbreeding, backcrossbreeding, recurrent backcross breeding or double haploid seedproduction. In a further embodiment, the invention relates to a methodfor self-fertilisation or intercrossing of dioecious plants wherein oneor both of the parent plants is a plant in which the functionalexpression of the dominant suppressor of gynoecium development isdisrupted or reduced. In a yet further embodiment, the invention relatesto a method to produce a plant, in which the functional expression ofthe dominant suppressor of gynoecium development is disrupted or reducedby inhibiting the expression of the GDS protein, preferably decreasingthe expression of the amino acid sequence depicted in SEQ ID NO: 2 or anortholog or functional homolog thereof. More particularly in thesemethods of the invention the disruption or reduction of the functionalexpression of the dominant suppressor of gynoecium development is causedby inhibiting expression of the GDS gene, preferably wherein the GDSgene comprises the sequence provided in SEQ ID NO:1 or is an ortholog, afunctional homolog or a functional fragment thereof. Preferably, themethods of the invention comprise a step of introducing a mutation inthe GDS gene to disrupt or reduce the functional expression of thedominant suppressor of gynoecium development. Consequently, it ispreferred that the above cited methods use a plant that comprises amutant GUS gene, preferably wherein the mutation is caused by a DNAreplacement. In a preferred embodiment the methods of the invention areperformed on a plant of the genus Asparagus, preferably Asparagusofficinalis.

Also part of the invention is a dioecious plant, preferably a plant ofthe genus Asparagus, more preferably a plant of the species Asparagusofficinalis, in which the expression of the dominant suppressor ofgynoecium development protein is disrupted or reduced. Preferably insaid plant the expression of the GDS gene is disrupted or reduced. In afurther preferred embodiment said plant has been subject to amutagenesis treatment, preferably wherein said treatment comprisesradiation with a radioactive element. Further preferred with respect tosaid plants is that it has been transformed or transfected with anucleotide sequence which is able to disrupt or reduce the expression ofsaid dominant suppressor of gynoecium development, preferably whereinsaid nucleotide sequence is homologous or partly homologous to asequence of the GDS gene, especially wherein said disruption orreduction of expression is reversible.

The invention also comprises a method to improve breeding in dioeciousplants comprising providing a plant in which the functional expressionof the dominant male stimulator is restored and introducing said plantin inbreeding, backcross breeding, recurrent backcross breeding ordouble haploid breeding techniques. In another embodiment, the inventioncomprises a method to improve breeding in dioecious plants comprising aplant wherein the lack of functional expression of the dominant malestimulator is complemented by a functional copy of the dominant malestimulator and introducing said plant in inbreeding, backcross breeding,recurrent backcross breeding or double haploid breeding techniques.Preferably in said methods the introduction of the dominant malestimulator is performed by inducing in a dioecious plant the expressionof a heterologous dominant male stimulator, preferably wherein saiddominant male stimulator is a TDF1 protein, preferably wherein said TDF1protein is the Asparagus officinalis TDF1 gene as depicted in SEQ ID NO:5 or an ortholog or functional homolog or functional fragment thereof,which functional fragment, preferably comprises at least the R2 and R3domains of the TDF1 protein or ortholog or functional homolog thereof.In a further preferred embodiment the gene encoding the dominant malestimulator is the Asparagus officinalis TDF1 gene as depicted in SEQ IDNO: 4 or an ortholog or functional homolog thereof or a fragment thereofcoding for a fragment of the TDF1 protein as defined above.

Further part of the invention is a method for self-fertilisation orintercrossing of dioecious plants wherein one or both of the parentplants is a plant in which the lack of functional expression of thedominant male stimulator is restored or complemented by a functionalcopy of the dominant male stimulator, preferably wherein said dominant,male stimulator is a TDF1 protein or ortholog or homolog thereof.

Also part of the invention is a method for in nitro androgenesis whereinthe plants used for providing anthers is a plant in which the lack offunctional expression of the dominant male stimulator is restored orcomplemented by a functional copy of the dominant male stimulator,preferably wherein said dominant male stimulator is a TDF1 protein orortholog or homolog thereof.

Also part of the invention is a protein that is able to suppressgynoecium development in asparagus plants comprising the amino acidsequence of SEQ ID NO: 2 or an ortholog or functional homolog thereof.Further encompassed in the present invention is a nucleic acid sequenceencoding said protein, wherein said nucleic acid sequence is the cDNAsequence as depicted in SEQ ID NO: 1 or the genomic sequence that can bederived from SEQ ID NO: 3.

Also part of the invention is a protein that is able to providemasculinization in a plant from a dioecious species, comprising theamino acid sequence of SEQ ID NO: 5 or an ortholog or functional homologthereof or a fragment thereof as defined above. Further encompassed inthe present invention is a nucleic acid sequence encoding the proteinaccording to claim 23, wherein said nucleic acid sequence is the cDNAsequence as depicted in SEQ ID NO: 4 or the fragment thereof that isable to code for the fragment as defined above.

Also part of the present invention is a hybrid plant of a dioeciousspecies obtained in a breeding scheme, preferably from an inbred plantproduced through one of the breeding methods according to the presentinvention.

Further part of the invention is a method to improve breeding indioecious plants comprising providing a feminized plant and introducingsaid plant in inbreeding, backcross breeding, recurrent backcrossbreeding or double haploid seed production.

Further comprised in the invention is a method to improve breeding indioecious plants comprising providing a defeminized plant andintroducing said plant in inbreeding, backcross breeding, recurrentbackcross breeding or double haploid seed production.

Further comprised in the invention is a method to improve breeding indioecious plants comprising providing a masculinized plant andintroducing said plant in inbreeding, backcross breeding, recurrentbackcross breeding or double haploid seed production.

Also comprised in the invention is a method to improve breeding indioecious plants comprising providing a demasculinized plant andintroducing said plant in inbreeding, backcross breeding, recurrentbackcross breeding or double haploid seed production.

LEGENDS TO THE FIGURES

FIG. 1-A

Example of scaffold 905 to illustrate the read coverage of reads ofDH00/094 (indicated as the ‘XX Female Resequence’ track) all of a suddendrops at position 104,688 (from 30× to zero) whereas the read coverageof the male DH00/086 (indicated as ‘YY male mapping’ track) remainshigh. This suggest that this region may represent the border between theautosomal part and the male specific part (MSY) of the sex chromosome.

FIG. 1-B

Example of scaffold 905 positions where the published markers Asp 1-T7and Asp2-SP6 are located. Note that Asp2-T6 is located very close topredicted gene Aof31527.1. Sequence reads are lacking for there-sequenced female (see the XX Female Resequence track) whereasabundant, reads occur for the re-sequenced male (indicated as ‘YY malemapping’ track). The lack of reads at position 312500 are the result ofunknown sequences NNNN present in mate pair reads.

FIG. 1-C

Example of Mlocus scaffold 4 positions where the published markersAsp1-T7 and Asp2-SP6 are located. Note that Asp2-T6 is located veryclose to predicted gene Aof0065.2. Sequence reads are lacking for there-sequenced female (see the XX Female Resequence track) whereasabundant reads occur for the re-sequenced male (indicated as ‘YY malemapping’ track). Note that this representation resembles that presentedfor scaffold 905 (FIG. 1-C) but that the orientation is reversed.Further note that where in scaffold 905 the second exon is broken up ontwo parts it shows up as a single exon in Mlocus_scaffold 4. Sangersequencing revealed that the Mlocus_scaffold 4 representation isaccurate for the second exon and thus a better representation comparedto Scaffold 905 which apparently comprises some minor assembly errors.

FIG. 2 (SEQ II) NO: 115)

Donor splice site intron2.

ML4 DUF247 at the position of the CDS2/Intron2 boundary. The EVM1prediction is shown directly above the plus-strand sequence and predictsa putative 5′-splice site indicated by the black bar: TG/GC. Two cDNAsequences derived from RNA isolated from flower buds of genotypeDH00/086 are below the minus-strand sequence indicated by CP35CR55_57and CR55CR57_57. The actual splice site is indicated by cDNA 5′-splicesite: GG/GT. The Cytosine at position 2795 has never been reported forplant donor splice sites. The Thymidine at position 2835 is 100%preserved.

FIG. 3A-C (SEQ ID NOS: 116.128)

Alternative cDNA sequences for the DUF247 gene based on differentanalyses of the genomic DNA of Asparagus officinalis.

FIG. 4

Short read alignment of G033 (shown as LIM_G033_Alignments) and K323(shown as LIM_K323_Alignments) against the Y-linked M-locus_scaffold4assembled scaffold annotated gene feature Aof000065.2. The track BGIgene annotations shows the FGENESH predicted exons in thick barsseparated by a thinner line to show the predicted intron. EVM showed anevidence based gene model (for description see text of EXAMPLE 1). Thedashed line border a region for which no reads are mapping for G033which indicates that this part of the DUF247 essentially is deleted.Arrows indicate clip-reads (see text) indicative of a border of theinsert.

FIG. 5 (SEQ ID NOS: 129.134)

A. Example of the two Sanger reads obtained from sequencing thehermaphrodite G033 using primer pair CN78/CN83 and a Sanger read of theWild type hybrid using primer pair CN59/CN70 as reference. For primerpairs see Primer Table 3.

B. Alignment of sequenced males, hermaphrodite 5375 and hermaphroditeG033 to show the intron position at which the sequence of G033 appearsdifferent compared to the other reads.

FIG. 6.

Cumulative number of plants that flowered since the first plants of thepedigree of the 3E a pseudo test cross: female 1800×selected F1(5375×1770) flowered (set as day 1). Solid lines curve representcumulative number of flowers from male plants and the dashed line showsthe cumulative number of flowers of hermaphrodite plants.

FIG. 7A

GENEVESTICATOR (www.genevestigator.org, NEBION AG, Zurich, Switzerland)experiment using all available gene expression data of 10 DUF247-likegenes of Arabidopsis across 10 developmental stages of Arabidopsislines. AT2G38540 is the unrelated Arabidopsis TDF1 gene. The Percent ofExpression Potential is displayed for each gene-stage combination withsix-group color indication.

FIG. 7B

GENEVESTICATOR (www.genevestigator.org, NEBION AG, Zurich, Switzerland)experiment using all available gene expression data of 9 color indicatedDUF247-like genes of Arabidopsis across 10 developmental stages ofArabidopsis lines. AT2G38540 is the unrelated Arabidopsis TDF1 gene. Thelevel of expression (signal intensity on Arabidopsis ATH1 genome array)is displayed for each gene-stage combination as LOW, MEDIUM or HIGH.

FIG. 7C

GENEVESTICATOR (www.genevestigator.org, NEBION AG, Zurich, Switzerland)experiment using all available gene expression data of 10 DUF247-likegenes of Arabidopsis across 127 anatomical parts of Arabidopsis lines.The Percent of Expression Potential is displayed for eachgene-anatomical part combination with six-group color indication. TheArabidopsis inflorescence data is shown in detail for flower organs, therelatively low values for gene expression in pistil data is highlighted.

FIG. 8A-E (portions of SER ID NOS: 137 and 139)

Overview of 2 gene predictions (dark) of FGenesh *ML4 DUF247 FG) and EVM(ML4 DUF247 EVM) and their respective coding sequences (CDS1, CDS2 forthe FG prediction and CDS1-CDS3 for EVM prediction). The middle barsrepresent the generic sequence and corresponding coding sequences asdetected by cDNA sequencing of mRNA from flower buds of DH MaleDH00/086. The 5-splice site of intron2 has shifted 42 bp upstream incomparison to the EVM prediction.

FIG. 9A

GENEVESTICATOR (www.genevestigator.org, NEBION AG, Zurich, Switzerland)experiment using gene expression data of 10 DUF247 domain containinggenes of Arabidopsis across selected anatomical parts of Arabidopsiswild type experiments. The selection included 4 datasets of young anddeveloped flower expression data. AT2G38540 is the unrelated ArabidopsisTDF1 gene. The Percent of Expression Potential is displayed for eachgene-anatomical part combination with six-group color indication. TheArabidopsis inflorescence data is shown in detail for flower organs, Therelatively low values for gene expression for 8 genes is highlighted.

FIG. 9B

Detailed view of the gene expression data for individual flowerexperiments in FIG. 9A.

FIG. 9C

Hierarchical Clustering (Pearson correlation indices) of anatomicalparts and Percent of Expression Potential indicated in FIG. 9A. Highcorrelation values for the 3 clusters of genes is indicated by thelength of lines in the respective correlation trees.

FIG. 10 (SEQ ID NOS: 135 and 136)

Sequence alignment of predicted coding sequence ML4 DUF247 EVM (depictedas EVM) and coding sequence for isoform ML4 DUF247 DH (depicted as DH)found in cDNA sequencing of DH00/086 flower buds derived total RNA. Seetable 6 for details of coding sequences.

FIG. 11

PCR products obtained for genoypes DH00/086 (the supermale of thereference genome sequence), hermaphrodite mutant G033 and supermale K323using primer pairs CN78/83) and CN78/CN84, respectively left and rightfrom the 100 bp size ladder which are diagnostic for the deletioninsertion event, thus unique sequences in the DUF247 gene ofhermaphrodite mutant G033. Note a unique and prominent PCR product forG033 whereas the other (male) samples show aspecific fingerprint-likepatterns.

FIG. 12

A: Phenotype of hermaphrodite G33 which shows full berry set. B: Flowersof three WT K323 plants (left hand side) next to three G033 flowers;note that the flowers of the hermaphrodite G033 shows longer styles, andbetter developed stigma's and larger fruits compared to the WT K323 maleplants. C: The difference in organ development of the G033 flower (left)and two flowers of all male hybrid K323 (the two at the right) next to aruler to allow estimation of size differences.

FIG. 13A-DH (SEQ ID NOS: 137.147)

Sanger reads obtained from PCR fragment sequencing using genoytpesDH00/086, 9M, 88M, K323, hermaphrodite 5375 and hermaphrodite G033 astemplate DNA and sequences of the scaffolds that are mentioned in thepresent application: |el|M-locus_scaffold4, Scaffold 905, Scaffold 3098,scaffold 10515.

FIG. 14

‘Examples of Flower Phenotypes’

Example of the female flower of the breeding line used as female in thetest-cross and two typical flowers that are representative for bothphenotypic classes that segregate in 861BC1d.

FIG. 15

“CHG Methylation and read coverage of K1036 vs DH00/086 and line 9 atScaffold_905” Per position CHG methylation levels are plotted as a bargraph for line K1036 (top graph) and for DH00/086 and line 9 (bottomgraph) for Scaffold_905 for position 49.815 to 51.249 (genome version2.0). Per position informative read coverage is plotted with crosses forK1036 and triangles for DH0086 and circles for line 9. Depending on thestrand of the CHG position, informative reads solely derive from eitherWatson or Crick strand. Note that for K1036 many CHG positions aremethylated (indicated by numerous bars) whereas the CHG methylatedpositions of DH00/086 and line 9 are very limited; note that there isonly a small number of bars to indicate methylation for DH00/086 andline 9 which means that at many other CHG positions the methylationlevels is equal to 0% (absence of bars).

FIG. 16.

Size distribution of PacBio long sequencing reads of 4.6× coverage ofAsparagus officinalis Male DH00/086.

FIG. 17.

BioNano contig BNG28 and its aligned AGS V2.0 scaffolds in the M locusregion. Arrows and primer codes show the location of primers tested inPCR to analyze the loss of hemizygosity or loss of heterozygosity whichis diagnostic for the size of the deletion caused by Cobalt 60 gammairradiation.

FIG. 18. (SEQ ID NO: 148)

TDF-like CDS of Asparagus officinalis. Exons are in capital font andshaded.

FIG. 19. (SEQ ID NO: 149)

The 276 AA translation of the MYB34-like asparagus ortholog of Defectivein Tapetal Development and Function 1 gene, homologous to ArabidopsisAT3G28470) and Oryza sativa osTDF1 (LOC_Os03g18480).

FIG. 20A-B. (SEQ ID NOS: 150-175)

tBLASTN result using ATH TDF1 as Query on a Database of AGS V2.0assembly. AGHS 2.0 scaffold 436 and 1220 have the highest identities.Yet AGS V2.0 scaffold 1220 has a lower identity in the first SANTdomain.

FIG. 21.

Fingerprinting using microsatellite markers (sat) and HRM markers toconfirm the authenticity of mutants found in particular hybrids. Mutantsgenotypes are shown together with control hybrids to which those mutantsbelong Several controls plants are shown to illustrate the variabilitythat is commonly observed for those markers. Parental alleles of thosehybrids are shown (when known).

FIG. 22

Images of flowers of mutants of hybrid K1150, K1129 and K323 obtainedafter Cobalt 60 irradiation and reference control flowers. Fordescriptions see Example 6 and Example 7.

FIG. 23A-B

Example to show the read depth for the scaffold parts. One harboring theAs-TDF1, and one harboring the GDS gene having a DUF247 domain. Notethat the read depth observed for the male-to-female mutants is lowerand/or that reads are absent indicative of the fact that the deletionoverlaps both the Y specific and the pseudo-autosomal region.

FIG. 24A-E

Represents the compositions of SEQ ID NOS: 1-6.

Definitions

In this description, unless indicated otherwise, the terms anddefinitions used herein are those used in (Mendelian) genetics, forwhich reference is made to M. W. Strickberger, Genetics, second Edition(1976), in particular pages 113.122 and 164-177. As mentioned therein,“gene” generally means an inherited factor that determines a biologicalcharacteristic of an organism (i.e. a plant), and an “allele” is anindividual gene in the gene pair present in a multiploid organism, suchas a diploid (asparagus) plant

A natural staminose plant defines as plant that naturally has one ormore functional anthers producing functional pollen.

The term staminose is defined as having a flower with one or morefunctional anther(s) producing functional pollen and excludes femaleplants. The term staminose may be similar to the term staminose as usedin Hendersons Dictionary of Biological terms, 11th edition p560 but maynot he similar to staminate which in the same handbook is described asflower containing stamens but no carpels.

Syngeneic is used to define genetically identical

Gynoecium refers to a collective term for the parts of a flower thatproduce ovules and ultimately develop into the fruit and seeds. Thegynoecium may consist of one or more separate pistils. A pistiltypically consists of an expanded basal portion called the ovary, anelongated section called a style and an apical structure that receivespollen called a stigma.

Gynoecium development refers to development of the gynoecium to produceovules and ultimately develop into fruit and seeds.

A natural female plant is a plant that produces only flowers that havefully developed female organs, such as a style and stigma and ovary thatallow fruit set and only produces rudimentary non-functional anthers ascan he found in nature because it naturally lacks a dominant suppressorof gynoecium development and naturally lacks a dominant gene conferringandroecium development.

Feminization or being feminized is defined as restoring or enhancing thegynoecium development of a plant by disrupting or decreasing thefunctional expression of the suppressor of gynoecium development (GDS)gene, its homolog(s) or ortholog(s), as defined in present document asthe result of human intervention.

The restored or enhanced development of the gynoecium in a feminizedplant may be determined by the skilled person in comparing it to asuitable reference plant, exposed to identical growing conditions, wherein case that a feminized plant produces less functional pollen comparedto the reference plants, it will be pollinated in such a way thatpollination itself will not limit fruit set. Said reference plant willhave the same ploidy level as the feminized plant, is not a female, andin said reference plant the functional expression of the suppressor ofgynoecium development (GDS) gene, its homolog(s) or ortholog(s),disclosed in present document has not been disrupted or decreased. Mostpreferably, the reference plant is syngeneic to the feminized plant thatis evaluated. Examples of preferred reference plants are syngeneicplants obtained by vegetative propagation of a plant to be feminized,prior to the human intervention targeting its GDS gene, preferably by ashort propagation step to avoid somaclonal variation which may rendertwo plants insufficiently syngeneic for a proper comparison. Anotherpreferred example of a suitable reference is (an average or member of) alarge number of full siblings resulting from a cross between two doubledhaploid parents, or true breeding (thus highly inbred) parents that arethe same parents of the hybrid, from which the feminized plant to beevaluated results, where said full siblings or any of their parents havenot been the subject of human intervention targeting a suppressor of agynoecium development (GDS) gene, its homolog(s) or ortholog(s). In casethe aforementioned preferred reference plants are not available, forexample in case the human intervention targeting the suppressor ofgynoecium development (GDS) gene was performed on a gamete the skilledperson may take a sufficiently large number of siblings, which are notfemale plants, where said sibling or any of their parents have not beenthe subject of such human intervention, as reference to the feminizedplant. If these siblings are not available or low in number the skilledperson can take as reference the direct male ancestor of the feminizedplant as reference plant, where said male ancestor has not been thesubject of human intervention targeting the suppressor of a gynoeciumdevelopment (GDS) gene, its homolog(s) or ortholog(s). To have said maleancestor available, the person of skill may vegetative propagate theancestor.

When reference plants are genetically variable, which would hamper thecomparison with highly syngeneic reference plants the skilled person maytest whether the zero hypothesis that the trait of restored or enhancedgynoecium development of the feminized plant segregates independently ofthe targeted GDS gene and/or its homolog(s) or ortholog(s) in a suitabletest cross population should be accepted or rejected. Fine mapping andphenotyping may then provide further clarification on the rol of the GDSgene in the feminization.

Restoring or enhancing gynoecium development as used in the definitionof feminization means that a plant, in which the gynoecium developmentis enhanced or restored, is better capable of producing berriescomprising viable seeds compared to a suitable reference plant.

Enhanced or restored gynoecium development may include an increase instyle length and more conspicuous stigma which can be measured orinferred by on a scale such as has been applied by Franken (1969, 1970)and Beeskov (1967), Enhancing or restoring gynoecium development on theaforementioned scales means that flower(s) of the feminized plant willobtain a higher score on said scales compared to the scores of thereference plant.

Defeminization or being defeminized is defined as disrupting ordecreasing gynoecium development, by restoring or increasing thefunctional expression of the suppressor of gynoecium development (GDS)gene, its homolog(s) or ortholog(s), as defined in present document, asthe result of human intervention.

The disrupted or decreased gynoecium development a defeminized plant maybe determined by the skilled person in comparing it to a suitablereference plant, exposed to identical growing conditions, where in casethat a reference plant produces less functional pollen compared to thedefeminized plants, it will be pollinated in such a way that pollinationitself will not limit fruit set. Said reference plant will have the sameploidy level as the defeminized plant, is a staminose plant, and in saidreference plant the functional expression of the suppressor of gynoeciumdevelopment (GDS) gene, its homolog(s) or ortholog(s), disclosed inpresent document has not been restored or increased. Most preferably,the reference plant is syngeneic to the feminized plant that isevaluated. Examples of preferred reference plants are syngeneic plantsobtained by vegetative propagation of a plant to be defeminized, priorto the human intervention resulting in restoring or increasingfunctional expression of a GDS gene, preferably by a short propagationstep to avoid somaclonal variation which may render two plantsinsufficiently syngeneic for a proper comparison. Another preferredexample of a suitable reference is (an average or member of) a largenumber of full siblings resulting from a cross between two doubledhaploid parents, or true breeding (thus highly inbred) parents that arethe same parents of the hybrid, from which the defeminized plant to beevaluated results, where said full siblings, or any of their parents,have not been the subject of human intervention resulting in restoringor increasing functional expression of a suppressor of a gynoeciumdevelopment (CDS) gene, its homolog(s) or ortholog(s) In case theaforementioned preferred reference plants are not available, for examplein case the human intervention restoring or increasing functionalexpression of a suppressor of gynoecium development (GDS) gene, wasperformed on a gamete the skilled person may take a sufficiently largenumber of siblings, which are staminose plants, where said siblings orany of their parents, have not been the subject of such humanintervention, as reference to the feminized plant. If these siblings arenot available or low in number the skilled person can take as referencethe staminose ancestor of the defeminized plant as reference plant,where said male ancestor has not been the subject of human interventionresulting in restoring or increasing functional expression of asuppressor of a gynoecium development (GDS) gene, its homolog(s) orortholog(s). To have said male ancestor available, the person of skillmay propagate the ancestor vegetatively.

When reference plants are genetically variable, which would hamper thecomparison with highly syngeneic reference plants skilled person maytest whether the zero hypothesis that the trait of disrupted ordecreased gynoecium development of the defeminized plant segregatesindependently of the restored or increased functional expression of theGDS gene and/or its homolog(s) or ortholog(s) in a suitable test crosspopulation should be accepted or rejected. Fine mapping and phenotypingmay then provide further clarification on the rol of the GDS gene in thedefeminization.

Disrupting or decreasing the gynoecium development as used in thedefinition of defeminization, means that a plant, that has saiddisrupted or decreased gynoecium development, is less capable ofproducing berries comprising viable seeds compared to a suitablereference plant.

Disrupting or decreasing gynoecium development, as used in thedefinition of defeminization may include a decrease in style length anda less conspicuous stigma which can be measured or inferred by on ascale such as has been applied by Franken (1969, 1970) and Beeskov(1967), Decreasing or disrupting gynoecium development on theaforementioned scales means that flower(s) of the defeminized plant willobtain a lower score on said scales compared to the scores of thereference plant.

The human intervention referred to in the present definition offeminization or defeminization includes any form of induced mutagenesis,be it by irradiation, chemical treatment or any other means ofmutagenesis. It also comprises any form of disruption (feminization) orrestoration (defeminization) of the gene or interference with thetranscription and translation of the gene. Examples for this are geneticmodification of the coding sequences, induction of splice variants,epigenetic changes due to methylation, inhibition of expression by RNAi,CRISPRi, anti-sense expression, modification of sites in thecis-regulatory elements of the gene, and the like. Also included iscrossing of plants that have a mutated gene with unmodified plants andselecting of offspring under guidance of marker assisted selection forthe presence of the mutated GDS gene.

Masculinization or being masculinized is defined as restoring orenhancing the androecium development by restoring or increasing thefunctional expression of the dominant male stimulator (e.g. AsOsTDF1),its homolog(s) or ortholog(s), as defined in present document isrestored or increased as the result of human intervention.

Restoring or enhancing androecium development in a masculinized plantmay be determined by the skilled person in comparing it to a suitablereference plant, exposed to identical growing conditions. Said referenceplant will have the same ploidy level as the masculinized plant, is nota natural staminose plant, and in said reference plant the functionalexpression of the male stimulator gene, its homolog(s) or ortholog(s),disclosed in the present document has not been restored or increased.Most preferably, the reference plant is syngeneic to the masculinizedplant that is evaluated. Examples of preferred reference plants aresyngeneic plants obtained by vegetative propagation of a plant to bemasculinized, prior to the human intervention resulting in restoring orincreasing the functional expression of a male stimulator gene,preferably by a short propagation step to avoid somaclonal variationwhich may render two plants insufficiently syngeneic for a propercomparison. In case the aforementioned preferred reference plants arenot available, for example in case the human intervention resulting inrestoring or increasing the functional expression of a male stimulatorgene, was performed on a gamete, the skilled person may take asufficiently large number of siblings, which are not staminose plants,where said siblings or any of their parents, have not been the subjectof such human intervention, as reference to the masculinized plant. Ifthese siblings are not available or low in number the skilled person cantake as reference the direct female ancestor of the masculinized plantas reference plant, where said female ancestor has not been the subjectof human intervention resulting in restoring or increasing thefunctional expression of its male stimulator gene, its homolog(s) orortholog(s). To have said female ancestor available, the person of skillmay propagate the ancestor vegetatively.

When reference plants are genetically variable, which would hamper thecomparison with highly syngeneic reference plants skilled person maytest whether the zero hypothesis that the trait of restored or enhancedandroecium development of the masculinized plant segregatesindependently of the restored or increased functional expression of amale stimulator and/or its homolog(s) or ortholog(s) in a suitable testcross population should be accepted or rejected. Fine mapping andphenotyping may then provide further clarification on the role of thetargeted male stimulator gene n the masculinization.

Restoring or enhancing androecium development as used in the definitionof masculinization means that a plant, obtaining said enhanced orrestored androecium development, is better capable of producingfunctional anthers comprising functional pollen compared to a suitablereference plant.

Enhancing or restoring androecium development may include an increase infilament length, a larger anther (thus increased in size), having atapetal (or tapetum) development comparable to a natural staminoseplant. Tapetal development comparable to a natural staminose asparagusplant means, that it will show no, or at least less tapetal degenerationcompared to what is typically observed in natural females.

Demasculinization or being demasculinized is defined as disrupting ordecreasing the androecium development of a plant by disruption ordecreasing the functional expression of the suppressor of the dominantmale stimulator (e.g. AsOsTDF1), its homolog(s) or ortholog(s), asdefined in present document is disrupted or decreased as the result ofhuman intervention.

Disrupting or decreasing androecium development in a demasculinizedplant may be determined by the skilled person in comparing it to asuitable reference plant, exposed to identical growing conditions. Saidreference plant will have the same ploidy level as the demasculinizedplant, is a staminose plant, and in said reference plant the functionalexpression of the male stimulator gene, its homolog(s) or ortholog(s),disclosed in present document has not been disrupted or decreased. Mostpreferably, the reference plant is truly syngeneic to the demasculinizedplant that is evaluated. Examples of preferred reference plants aresyngeneic plants obtained by vegetative propagation of a plant to bedemasculinized, prior to the human intervention targeting the malestimulator gene, preferably by a short propagation step to avoidsomaclonal variation which may render two plants insufficientlysyngeneic for a proper comparison. Another preferred example of asuitable reference is (an average or member of) a large number of fullsiblings resulting from a cross between two doubled haploid parents, ortrue breeding (thus highly inbred) parents that are the same parents ofthe hybrid, from which the demasculinized plant to be evaluated results,where said full siblings, or any of their parents, have not been thesubject of human intervention targeting a male stimulator gene, itshomolog(s) or ortholog(s) In case the aforementioned preferred referenceplants are not available, for example in case the human interventiontargeting a male stimulator gene; was performed on a gamete the skilledperson may take a sufficiently large number of siblings, which arestaminose plants, where said siblings or any of their parents, have notbeen the subject of such human intervention, as reference to thedemasculinized plant. If these siblings are not available or low innumber the skilled person can take as reference the direct male orstaminose ancestor of the demasculinized plant as reference plant, wheresaid staminose ancestor has not been the subject of human interventiontargeting its male stimulator gene, its homolog(s) or ortholog(s). Tohave said staminose ancestor available, the person of skill mayvegetative propagate the ancestor.

When reference plants are genetically variable, which would hamper thecomparison with highly syngeneic reference plants skilled person maytest whether the zero hypothesis that the trait of disrupted ordecreased androecium development of the demasculinized plant segregatesindependently of the targeted male stimulator and/or its homolog(s) orortholog(s) in a suitable test cross population should be accepted orrejected. Fine mapping and phenotyping may then provide furtherclarification on the rol of the targeted male stimulator gene in thedemasculinization.

Decreasing or disrupting androecium development as used in thedefinition of demasculinization means that a plant, obtaining saiddisrupted or decreased development is less capable of producingfunctional anthers comprising functional pollen compared to a suitablereference plant.

Decreasing or disrupting androecium development may include a decreasein filament length, a smaller anther (thus decreased in size), having atapetal (or tapetum) development comparable to a natural female plantsuch as showing tapetal development comparable to a natural female plantmeans, that it will show equal absence of tapetal development astypically observed in female plants or at least less tapetal developmentcompared to what is typically observed in staminose plants

The human intervention referred to in the present definition ofmasculinization and demasculinization includes any form of inducedmutagenesis, be it by irradiation, chemical treatment or any other meansof mutagenesis. Tt also comprises any form of restoration(masculinization) or disruption (demasculinization) of the gene orinterference with the transcription and translation of the gene.Examples for this are genetic modification of the coding sequences,induction of splice variants, epigenetic changes due to methylation,inhibition of expression by RNAi, CRISPRi, anti-sense expression, ormodification of cis-regulatory elements of the gene and the like. Alsoincluded is crossing of plants that have a mutated gene with unmodifiedplants and selecting of offspring under guidance of marker assistedselection for the presence of the mutated male stimulator gene.

Male ancestor is defined as a staminose plant capable of producingfunctional anthers belonging to pedigree of a plant from which thelatter plant is derived, which may include vegative propagation of theancestor its somatic cells or somatic tissue from which a plant isderived.

A pedigree, is a list of the ancestors from which a plant has descended

Suppression of gynoecium development or inhibition of gynoeciumdevelopment is defined as the phenomenon, typically observed in male andandromonoecius (thus different from hermaphrodite) or neuter plants thata dominant suppressor gene hampers the development of the gynoecium.Commonly, suppression of gynoecium development is not observed innatural females, or natural hermaphrodites which produce many berries,comprising viable seeds, and should produce berries from all of theirflowers, provided that those plants are growing under optimal conditionsand provided that those plants can be fertilized by viable pollen and donot suffer from inbreeding depression or mutations that may causereduced fitness affecting fruit set. Plant hypothesized to exhibitsuppression of gynoecium development do not set fruit from their flowersor not from all of their flowers, even when they grow under optimalconditions and can he fertilized by viable pollen and do not suffer frominbreeding depression or mutations, that may cause reduced fitnessaffecting fruit set. Apart from a decreased capability to produceberries, comprising viable seeds, plants which show suppression ofgynoecium development are expected to exhibit a significantly decreasein style length and/or a less conspicuous stigma which may be measuredor inferred using a scale such as has been applied by Franken (1969,1970) and Beeskov (1967). According to the scale of Beeskov (1967), aplant showing suppression of gynoecium development is expected to haveflowers that will be classified with a score less than IV, preferablyless than Ill preferably less than II preferably equal to I. Accordingto the scale of Franken (1969, 1970) a plant showing suppression ofgynoecium development is expected to have flowers that will beclassified with a score less than 5, preferably less than 4 preferablyless than 3, preferably less than 2, preferably equal to 1. Suppressionof gynoecium development is expected to be the result of functionalexpression of a gynoecium development suppressor GDS gene, that ishomologous to sequences provided in present document. That a dominantsuppressor gene is active in a plant may be tested by phenotyping thetest-cross progeny of a plant and rejecting the hypothesis that adisrupted gynoecium development phenotype and markers linked to theM-locus such as those that are described in the art segregateindependently.

Androecium refers to a collective term for the stamens of a flower,where a stamen typically consists of a stalk called the filament and ananther which contains microsporangia in which pollen grains develop frommacrospores.

TDF1 marker assisted selection is defined as marker assisted selectionhaving an aim to introduce any mutation that induces masculinization ordemasculinzation or that is guided by information based on assays (suchas but not limited to Sanger Sequencing, CAPS markers analysis, highresolution melting curve marker analysis, Taqman assays, Kasp assays,etc.) designed to elucidate sequence information of the TDF1 gene, itshomologs or orthologs disclosed in present document into a plantpedigree. TDF1 marker assisted selection may also include usinginformation, designed to elucidate sequence information of the TDF1gene, its homologs or orthologs of a parental plant, followingintroduction of a desired TDF1 gene allele in a pedigree, where othermarkers than those targeting the TDF1 gene are used that aresufficiently linked, preferably within 20cM, more preferably within10cM, more preferably within 5cM, more preferably within 1cM, to thedesired TDF gene allele.

GDS marker assisted selection is defined as marker assisted selectionhaving an aim to introduce any mutation(s) that induces feminization orthat is guided by information that may be based on sequencing or assays(such as but not limited, CAPS markers analysis, high resolution meltingcurve marker analysis, Taqman assays Kasp assays etc) designed toelucidate sequence information of the GDS gene, its homologs ororthologs disclosed in present document into a plant pedigree. GDSmarker assisted selection may also include using information, designedto elucidate sequence information of the GDS gene, its homologs ororthologs of a parental plant, following introduction of a desired GDSgene allele in a pedigree, where other markers than those targeting theGDS gene are used that are sufficiently linked, preferably within 20cM,more preferably within 10cM, more preferably within 5cM, more preferablywithin 1cM, to the desired GDS gene allele.

Mutagenesis or mutagenesis treatment is defined as enabling, preferablyenhancing, the process by which the genetic information of an organismis changed in a stable manner, resulting in a mutation that is achievedexperimentally, thus which is different from a mutation arisingspontaneously in nature, by applying a non-natural doses of irradiationor unnatural exposure to a mutagenic agent.

The dominant male stimulator (gene) is a gene linked to, or present atthe M locus that confers the development in staminose plants or geneproduct derived from this gene. This dominant male stimulator (alsoindicated as stimulator of androecium development or stimulator ofanther development) is a protein that is encoded by a gene that isidentical to or a homolog or ortholog of the TDF1 (defective in TapetalDevelopment and Function) gene, which is found in Arabidopsis AT3G28470and in rice (osTDF1, LOC_Os03g18480). The sequences of the orthologousgene in Asparagus officinalis, AsOsTDF1, are provided by SEQ ID NO:4,SEQ ID NO:5, and SEQ ID NO:6.

For several reasons the procedure of somatic embryogenesis described inthe section “protoplast culture’ of Maeda et. al (2005) includingreference to a method of Kunitake and Mii (1990) and transplanting theplants obtained by said somatic embryogenesis to a field is not includedas an embodiment of human intervention of enhancing physicalcharacteristics of the gynoecium. As has been discussed in the presentdocument reviewing the literature, the work of Meada (2005) providesinsufficient teaching to the skilled person that a sex converted planthas been obtained from somatic embryogenesis and that this would providea workable method. Although it can not be fully excluded thatsufficiently proven sex converted plants may ever be generated by theembryoculture followed by transplanting as applied by Maeda et al(2005), the authors of the present invention have no problem to excludethe method described by Maeda et al (2005) as human intervention used todefine feminization in the present document. The skilled person willunderstand that any method of human intervention which extends tosomatic embryogenesis and transplanting described by Maeda et al (2005)thus includes additional steps. Preferably said steps would comprisesthe application of mutagenesis or GDS marker assisted selection, eitherbefore or after somatic embryogenesis, but excluding conventionalcrossing to generate viable offspring as the sole additional humanintervention. In such a way it might be possible to obtain a feminizedplant, which then would be a different method.

A natural male asparagus plant is defined as a plant that is capable ofproducing flowers with fully developed anthers as can be found in naturebecause it has at least one natural functional copy of the dominantasparagus gene homologous to defective in tapetum development andFunction 1 (TDF1).

A plant is called “homozygous” for a gene when it contains the samealleles of said gene, and “heterozygous” for a gene when it contains twodifferent alleles of said gene. The use of capital letters indicates adominant (form of a) gene and the use of small letters denotes arecessive gene: “XX” therefore denotes a homozygote dominant genotypefor gene or property X; “Xx” or “xX” denote heterozygote genotypes; and“xx” denotes a homozygote recessive genotype. As is commonly known, onlythe homozygote recessive genotype will generally provide thecorresponding recessive phenotype (i.e. lead to a plant that shows theproperty or trait “x”) whereas the heterozygotic and homozygote dominantgenotypes will generally provide the corresponding dominant phenotype(i.e. lead to a plant that shows the property or trait “X”), unlessother genes and/or factors such as multiple alleles, suppressors,codominance etc. (also) play a role in determining the phenotype. Aplants is called “hemizygous”, when it has only one member of achromosome pair or chromosome segment rather than the usual two; morespecifically in the present description the term hemizygous refers tocertain Y linked genes, thus in the male chromosome, in a way that amale plant has a chromosome segment that is lacking in females.

As used herein, the term “plant” includes the whole plant or any partsor derivatives thereof, such as plant cells, plant protoplasts, plantcell tissue cultures from which plants (e.g. Asparagus officinalisplants) can be regenerated, plant calli, plant cell clumps, and plantcells that are intact in plants, or parts of plants, such as embryos,pollen, ovules, fruit (e.g. harvested tomatoes), flowers, leaves, seeds,roots, root tips and the like.

An ortholog or orthologous gene according to the present invention wouldbe a gene that has evolved divergently between species or evenvarieties. This means that an ortholog of the GDS gene as defined hereinwould mean any gene in a species different from the species or varietyfrom which the GDS sequence in this application has been derived andhaving evolved from the same ancestral sequence. It will be recognizedthat in most, if not all, of the cases of orthologous genes, thefunction of said gene is maintained. In this sense, automatically, anortholog of the GDS gene as specified herein has the same function asdescribed in the application for the GDS gene of Asparagus officinalis.Orthologs may share a large degree of homology, but not necessarily.Often orthologous genes in a different species are found in the similargenetic environment, i.e. clustered within a gene cluster that can besaid to be orthologous for most of the genes present in the cluster.

A homolog or homologous sequence according to the present invention is asequence which has a high level of sequence identity with the sequenceof which it is said to be a homolog. A high sequence identity or highhomology in this respect means for a nucleic acid sequence that twohomologous sequences would selectively hybridize, under selectivehybridization conditions, to each other. A homologous nucleic acid issaid to be a functional homolog or a functional homologous sequence ifit would code for an amino acid sequence which has a biological functionsimilar to the function of the protein encoded by the gene of which itis said to be homologous with.

In this sense, the definition of high sequence identity in the presentinvention includes nucleotide sequences which have a percentage ofidentity related to the sequences with which they are said to behomologous of 65% to 95%. Thus, for example, the percentage of identitycan be at least, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. Sequence identityon basis of nucleotide sequences can be calculated by using the BLASTNcomputer program (which is publicly available, for instance through theNational Center for Biotechnological Information, accessible via theinternet on http://www.ncbi.nlm.nih.gov/) using the default settings of11 for wordlength (W), 10 for expectation (E), 5 as reward score for apair of matching residues (M), −4 as penalty score for mismatches (N)and a cutoff of 100. Alternatively, the homology can be calculated onbasis of the amino acid sequence of the protein encoded by saidnucleotide sequences. For amino acids, the sequence identity can becalculated through the BLASTP computer program (also available throughhttp://www.ncbi.nlm.nih.gov/). On the amino acid level functionalhomologs are defined as amino acid sequences having a sequence identityof at least 50%, preferably at least 55%, more preferably at least 60%,more preferably at least 70%, more preferably at least 80%, morepreferably at least 90%, more preferably at least 95% to the amino acidsequence of said protein. Functional homologs or orthologs of proteinsare defined as having a biological function similar to the protein withwhich they are said to be homologous or orthologous to. A nucleic acidsequence encoding an amino acid sequence can have many variants. Becauseof the nature of the genetic code there are different triplets ofnucleotides that would translate into one and the same amino acid. It isto he understood that a nucleic acid encoding a protein may varyconsiderably without resulting in a different amino acid sequence. Sucha wobble of the genetic code may not influence the homology level of twonucleic acid sequences encoding a homologous or orthologous protein: ifthe encoded protein is deemed to be highly homologous or orthologousaccording to the definition as used herein, then also the encodingnucleic acids should be considered to be highly homologous.

DETAILED DESCRIPTION OF THE INVENTION

It is a first object of the present invention to provide a method forchanging the gender or sex of a plant by changing the expression of afemale suppressor gene and/or changing the expression of a gene thatenables androecium development. Further object is an alternative methodfor self fertilization or intercrossing of dioecious plants, preferablyasparagus plants, by using a loss of function of a female suppressorgene and/or by providing a gene that enables androecium development.

It is a second object to provide a technical teaching on how ‘feminizedplants’ a hermaphrodite, or partly hermaphrodite or andromonoeciusplant, or a female plant preferably from the genus Asparagus can beobtained unequivocally, in a way that differs from those known in theart.

A third object is to provide technical teaching on how female plantscould be masculinized into male plants that have a functionalandroecium.

It was established in the present invention by carefully designedtestcrosses that monogenic recessive sex linked inheritance of afeminized plant, in particular of a hermaphrodite phenotype exists.Further, this sex linked dominant repressor of gynoecium development wasidentified. Characterization of this gene revealed that this is a DUF247 domain containing gene. Ten mutants, having either a hermaphroditeor female phenotype were found, all of them containing a differentmutation relating to the expression of this gene, designated in thepresent invention as the GDS gene. Some mutants were found to also lackthe expression of a functional TDF1 gene (the Defective in TapetalDevelopment and Function 1 gene, homologous to Arabidopsis AT3G28470)and Oryza sativa osTDF1 (LOC_Os03g18480) which changed their phenotypefrom male to female.

This existence of a monogenic recessively inherited sex linkedhermaphrodite phenotype, tested for its segregation in pedigrees ofthree independent mutants, suggests that the gene conferringfeminization is the dominant female suppressor in asparagus that hasbeen predicted for dioecious species in general by evolutionarybiologists (Westergaard, 1958, Charlesworth and Charlesworth, 1978) butwhich never been convincingly proven to exist in asparagus. The presentinvention teaches that such a female suppressor indeed exists inasparagus and can be manipulated in such a way that it looses its femalesuppressing ability and converts an originally strictly male plant intoa plant that has perfect flowers that can be self-fertilized and/orcrossed to another male plant or that, in case the manipulation of thefemale suppressor is lost together with the male stimulator, anoriginally male plants can be converted into a female plant.Furthermore, the present invention describes that an allele of thefemale suppressor that has lost its female suppressing ability can beintroduced/introgressed together with a genetically linked male (pollen)fertility in other plants to create new hermaphrodite plants. Thepresent invention describes a method that is different from existingmeans of self fertilization or crossing to other male plants such asusing andromonoecious plants or hermaphrodite plants for which othergenetic models have been described—or at least have been geneticallymore complex—than the simple monogenic recessive inheritance that isexploited in the present invention. Further, the invention comprises amethod to provide such a plant, also wherein said plant only temporarilyexpresses this phenotype.

In addition the invention discloses a method to change female plantsinto male plants which should be accomplished by introducing afunctional copy or the gene product of a TDF1 gene.

The skilled person will understand that switching the female suppressoron and off or, more subtly, partly enhancing or reducing suppression ofgynoecium development is used in its broadest interpretation. Enablingor enhancing the suppression of gynoecium development can be the resultof providing a functional copy of the gene that confers suppression ofgynoecium development. ‘Switching off’, disabling or reducing thesuppression of gynoecium development would include any method to reducethe expression or functionality of gene conferring suppression ofgynoecium development. The skilled person will also understand thatapplication of switching the gene conferring suppression of gynoeciumdevelopment on and off or reducing and enhancing suppression of saidgene is not limited to only providing plants that carry functionalanthers. If the gene conferring suppression of gynoecium development ofa male plant is (partly) switched off (e.g. reduced in functionalexpression) it indeed may result in (more) andromonoecious plants orhermaphrodite plants. However, reduction of suppression of gynoeciumdevelopment may also coincide with the absence or reduction of antherfunctionality such as, but not limited to, the event that both thesuppressor of gynoecium development and the stimulator of antherdevelopment are jointly disrupted by a single deletion. In such a case,a male or andromonoecious plant is changed into a female plant and suchevents (in which changing both the dominant suppressor of gynoeciumdevelopment coincides with disruption of the stimulator of androeciumdevelopment) are also included in the present invention as methods tocontrol the functionality of the suppressor of gynoecium development.

In this context it is understood that the term gynoecium developmentsuppressor-gene (GDS gene) or -allele as used herein refers to an allelehaving the sequence depicted in SEQ

ID No: 1 or functional homologs or orthologs thereof. A preferredexample of such a gene or -allele is the particular Asparagus DUF247domain containing gene of which the cDNA is provided in SEQ ID NO:1.Accordingly, part of the invention are all nucleic acid sequences thatare able to encode a protein that is an ortholog of or functionalhomologous with the amino acid sequence encoded by the nucleotidesequence of SEQ ID NO: 1

It has been shown herein that loss of function of this gene lifts theinhibition of gynoecium development. Loss of function or reducedfunction of the GDS gene is determined quantitatively by detecting thatthe number of berries and seeds produced on a plant is increasedrelative to plants of the same pedigree generation or previousgenerations of the pedigree to which said plant belongs. Such a loss offunction will generally be caused by a mutation that is novel comparedto previous generations of that pedigree. A mutant GDS gene or -alleleas used herein may thus refer to any loss of function of the GDS genethat results in producing or contributing to the phenotypes of theinvention One mutant is the GDS-deletion-insertion allele, obtained as aresult from gamma irradiation treatment, described herein that has adeletion-insertion event starting at the 1820th nucleotide ofScaffoldMlocus4 (Genome version V1.1). that is inferred to cause theabsence of coding information after the nucleotide 567 of SEQ ID NO:1Another mutant, described herein, is the GDS-deletion-allele that has athymine (single base pair) deletion at the 3′ end of the first exon ofthe GDS gene, which is a deletion of a thymine that corresponds to adeletion at position 527 of SEQ ID NO:1, which will lead to a readingframe shift.

Another mutation, described herein, is a GDS epi-allele which resultsfrom hypermethylation wherein said methylation covers the firstpredicted exon, the first predicted intron and partly overlaps thesecond predicted exon 2 of the GDS gene. Said methylation is notably(but not strictly) CHG methylation (spanning nucleic acids 309762-308323of scaffold 905 (Genome version V1.1) or 1053-2492 or ScaffoldMlocus4(Genome Version 1.1). The observed differential CHG methylation of theepi-allele, will overlap with SEQ ID NO:1 at nucleotides in the intervalfrom the 5th to the −859th nucleotide. Yet another mutant, describedherein (K57561 is a GDS gene allele that is characterized by a cytosineto an adenine change at position 684 of SEQ ID NO:1. that leads to aproline into a threonine amino-acid change (Pro→Thr)

Another mutation, described herein, is a GDS allele [K4381 that ischaracterized by cytosine to an adenine change at position 166 of SEQ IDNO:1.

Another mutation, described herein, is a GDS allele [K1150], resultingfrom gamma irradiation treatment, characterized by an adenine to guaninemutation at a position that corresponds to position 1193 in SEQ ID NO:1,which leads to an asparagine (N) to serine (S) amino acid change.

Another mutation described herein is a GDS allele [K1129.300-8] is anadenine to thymine change identical to nucleotide position 1160 of SEQID NO:3. This adenine to thymine change is separated by 665 nucleotidesfrom the adenine of the first predicted start codon of the GDS geneThree similar mutations, described herein, are three independentlyobtained non-natural GDS null-alleles, where the GDS gene has beenentirely deleted (in the present case as the result of gamma irradiationtreatment) which was inferred from the loss of genetic marker allelesand sequences n.

The GDS gene is herein understood as a gene comprising a Domain ofUnknown Function 247 in its protein sequence that may belong to a groupof proteins which in dioecious asparagus species, represses pistildevelopment and fruiting. Preferred examples of said GDS gene is theAsparagus DUF247 domain containing gene as described herein. However,the invention also comprises functional homologs and/or orthologs ofthis GDS gene.

Also used in the present specification is the term “dominant suppressorof gynoecium development”. This term more clearly explains the functionof the female suppressor GDS gene but for the remainder should he deemedto be identical to this term.

The female suppressor gene that suppresses gynoecium development mayalso be introduced in other plants, for instance to provide femalesterility in in case fruit set is undesirable.

The dioecious plant of the invention is preferably of the genusAsparagus, more preferably of the species Asparagus officinalis.However, the invention is also contemplated for other dioecious plantssuch as the crops Cannabis, Dioscoreophyllum volkensii, Humulus,Pistacia, Taxus and Valerians.

Asparagus is a genus in the plant family Asparagaceae, subfamilyAsparagoideae. It comprises up to 300 species. Most are evergreenlong-lived perennial plants growing from the understory as lianas,bushes or climbing plants. The best-known species is the edibleAsparagus officinalis, commonly referred to as just asparagus. It is theaim of the present invention to change the gender of an Asparagus plantor to cross and select an Asparagus plant belonging to the subgenusAsparagus (see Norup et al 2015 and the subgenus Asparagus clade intheir FIG. 3) for species that usually are dioecious, such as but notlimited to A. aphyllus, A. stipularis, A. filicinus, A. schoberoides, A.kiusianus, A. oligoclonos, A. maritimus, A. inderiensis, A. officinalis,or A. cochinchinensis or A. prostratus or are usually gynodioecius, suchas but not limited to, A. plocamoides, A. altissimus, A nesiotes and Aacutifolius. In case the text refers to asparagus or Asparagus plants orasparagus plants, at least all of the above Asparagus species or anyasparagus plant belonging to the genus Asparagus to be used in breedingare included.

Nucleic acid sequences or fragments comprising suppressor of gynoeciumdevelopment (GDS) genes and alleles

and nucleic acid sequences or fragments comprising GDS genes and allelesmay also be defined by their capability to “hybridise” with the GDS asdescribed above, and more particularly the sequence provided in SEQ IDNO: 1 or SEQ ID NO:3 or splice variants of said gene, preferably undermoderate, or more preferably under stringent hybridisation conditions.“Stringent hybridisation conditions” are herein defined as conditionsthat allow a nucleic acid sequence of at least about 25, preferablyabout 50 nucleotides, 75 or 100 and most preferably of about 200 or morenucleotides, to hybridise at a temperature of about 65° C. in a solutioncomprising about 1M salt, preferably 6×SSC or any other solution havinga comparable ionic strength, and washing at 65° C. in a solutioncomprising about 0.1 M salt, or less, preferably 0,2×SSC or any othersolution having a comparable ionic strength. Preferably, thehybridisation is performed overnight, i.e. at least for 10 hours andpreferably washing is performed for at least one hour with at least twochanges of the washing solution. These conditions will usually allow thespecific hybridisation of sequences having about 90% or more sequenceidentity.

“Moderate hybridization conditions” are herein defined as conditionsthat allow a nucleic acid sequences of at least 50 nucleotides,preferably of about 200 or more nucleotides, to hybridise at, atemperature of about 45° C. in a solution comprising about 1 M salt,preferably 6×SSC or any other solution having a comparable ionicstrength, and washing at room temperature in a solution comprising about1M salt, preferably 6×SSC or any other solution having a comparableionic strength. Preferably, the hybridisation is performed overnight,i.e. at least for 10 hours, and preferably washing is performed for atleast one hour with at least two changes of the washing solution.

These conditions will usually allow the specific hybridisation ofsequences having up to 50% sequence identity. The person skilled in theart will be able to modify these hybridisation conditions in order tospecifically identify sequences varying in identity between 50% and 90%.

An important embodiment of the present invention is a method to improvebreeding in dioecious plants comprising providing a plant in which thefunctional expression of the dominant suppressor of gynoeciumdevelopment is disrupted or reduced and introducing said plant in [1]inbreeding, [2] backcross breeding or recurrent backcross breeding or[3] double haploid breeding techniques. As has been indicated in thebackground section, the breeding of dioecious plants is hampered becauseof limitations in the use of self-fertilization, backcrossing and seedpropagation. Provision of a plant of the invention in which expressionof the GDSgene is disrupted or hampered solves this problem, because itenables the development of a hermaphrodite or partly hermaphroditeplant, which can be used to generate a true-breeding parent line.

In a particular embodiment of the invention, ‘the hermaphrodite trait’described in present invention is used in breeding of dioecious plants,more preferably Asparagus plants in order to create inbred lines.

Essentially, creating one or more inbred line(s) according to thepresent invention comprises the steps of:

[1] Creating a novel hermaphrodite plant in which the functionalexpression of the dominant suppressor of gynoecium development isdisrupted or reduced, which results in plants having both a functionalgynoecium of a plant and a functional androecium, hereafter alsoreferred to as a plant that has a ‘hermaphrodite trait’. How such anovel hermaphrodite plant in which the functional expression of thedominant suppressor of gynoecium development is disrupted or reduced canbe created, is described further hereinbelow.

[2] Preparing a novel hermaphrodite plant by preparing a hybrid plant,comprising the ‘hermaphrodite trait’, by at least one cross in which afirst plant comprising the ‘hermaphrodite trait’ is crossed with asecond plant.

[3] Facilitating the self-fertilization of the plant obtained in step[1] or step [2], and selecting from the progeny thereof one or morepreferred plant(s).

[4] Optionally repeating the step of self-fertilization of the plant(s)obtained in step [3] one or more times and selecting from the progenythereof one or more preferred plant(s)

[5] Optionally changing the gender of a plant comprising the‘hermaphrodite trait’ obtained in step [3] or [4] into a male plant bysufficiently restoring the function or expression of the dominantsuppressor of gynoecium development

In one particular embodiment of the invention said novel hermaphroditeplant of step [2] is created by a first plant comprising said‘hermaphrodite trait’ with a second plant of female gender, andselecting from the progeny thereof plants comprising said ‘hermaphroditetrait’;

In another embodiment of the invention, said novel hermaphrodite plantof step [2] is created by crossing a first plant comprising said‘hermaphrodite trait’ with a second plant comprising said ‘hermaphroditetrait’, and selecting from the progeny thereof a plant comprising said‘hermaphrodite trait’

In yet another embodiment of the invention said novel hermaphroditeplant of step [2] is created by crossing a first plant comprising said‘hermaphrodite trait’ with a second plant of male gender that is nothomozygous for the dominant suppressor of gynoecium development, andselecting from the progeny thereof a plant comprising said‘hermaphrodite trait’

In another embodiment of the invention, said novel hermaphrodite plantof step [2] is created by a first step (a) in which a first plantcomprising said ‘hermaphrodite trait’ is crossed with a second plant ofmale gender that is homozygous or heterozygous for the dominantsuppressor of female development, and selecting from the progeny thereofa male plant that will he able to transfer the ‘hermaphrodite trait’ toa next generation, followed by a second step (b) in which the male plantobtained in step (a) is crossed as a first plant with a second plantthat is not homozygous for the dominant suppressor of gynoeciumdevelopment, and selecting from the progeny thereof a plant comprisingsaid ‘hermaphrodite trait.

Related to this first embodiment, is the provision of a female plant ofthe invention in which expression of both the GDS and TDF1 genes arejointly disrupted or hampered. Such a plant can aid in solving thisproblem, because it enables crossing this particular female plant aspistillate parent with a plant from which said female plant was derived.but which plant still contains both the GDS and TDF1 genes. Such a crossthen essentially is a cross that can be used to generate a true-breedingparent line. This possibility relating to the embodiment discussed aboveis mentioned to clarify that a plant in which the GDS gene is disruptedor hampered may enable the development of a hermaphrodite or partlyhermaphrodite plant, but in particular cases extends to the developmentof a female plant.

In yet another embodiment of the invention the hermaphrodite trait isexploited in back-cross breeding. In particular the present inventionprovides a method for introducing a ‘genetic trait’ into the geneticbackground of a super-male plant, to provide an syngeneic super-maleplant, where a super-male is defined as a plant that will not be able toprovide female plants in its direct offspring by fertilizing a femaleplant. By the present invention super-male plants can be obtained thatare highly syngeneic because of the ability to make direct crossesbetween a first degree relative of the super-male and the super-maleitself. Accordingly, the present invention provides a method that allowsa direct cross of a first-degree relative and its super-male parent toobtain offspring by said cross. This is achieved by providing a method,comprising the steps of:

[1] Preparing F1 hybrid plant progeny as a first step to introduce a‘genetic trait’ (i.e. a trait of interest) into the genetic backgroundof a super-male plant by crossing a first plant comprising said ‘genetictrait’ with a second plant, which is a super male, and selecting fromthe progeny thereof a plant that is capable to transfer the ‘genetictrait’ to a next generation

The skilled person may appreciate that in step [1] a first plant that isable to transfer the ‘genetic trait’ into the genetic background of asuper-male plant can be of any gender. However, in case said first plantis of male gender, either the first plant, or the second plant or bothplants, thus at least a single plant, used in the cross of step [1] mustbe capable of seed production. Such a plant capable of seed productionshould be feminized. Either such a feminized plant is a‘male-to-hermaphrodite-transgender or ‘male-to-andromonoecioustransgender’. Such a plant thus may be the result of disrupting thefunction of the dominant suppressor of gynoecium development or reducingthe expression of the dominant suppressor of gynoecium development or itis a male-to-female-transgender as the result of disrupting the functionof the dominant suppressor of gynoecium development or reducing theexpression of the dominant suppressor of gynoecium development of aplant in which also the stimulator of androecium development has beendisrupted or reduced in its expression

The skilled person will recognize that the present invention provides amethod to make an F1 hybrid by crossing a male plant that is able totransfer a ‘genetic trait’ directly to a super-male plant, whichhitherto has been impossible in the art, unless either the first plantor the second plant or both plants used in step [1] express naturalhermaphroditism or andromonoecy that differs from the feminizationaccording to the present invention. The skilled person will recognizethat it is not necessary to specifically describe whether or not a plantis feminized-in step [1] as this first step described that ‘progeny’must be obtained from which ‘a plant, that is principally capable totransfer the ‘genetic trait’ to a next generation, can be selected’.However the skilled person will appreciate the ability, thus flexibilityto use a male plant that is able to transfer the ‘genetic trait’ asfirst plant in step [1].

[2] The second step (BC1 or Back-Cross 1) is crossing the hybridobtained in step [1] with a second plant that is the same or has asimilar genotype as the super-male used in step [1], where either thefirst plant, or the second plant or both plants, thus at least a singleplant of this crossing of step [2], is feminized as the result ofdisrupting the function of the dominant suppressor of gynoeciumdevelopment or reducing the expression of the dominant suppressor ofgynoecium development, preferably transiently, and selecting from theBC1 progeny thereof a plant that is principally capable to transfer the‘genetic trait’ to a next generation, and has a functional androecium.

[3] Optionally repeating step [2] one or ‘n’ times to warrant that thehybrid obtained in step [2] is sufficiently syngeneic to the super-maleplant first used in step [1] and selecting from the progeny thereof aBC2 or BCn plant that is principally capable to transfer the ‘genetictrait’ to a next generation and has a functional androecium.

[4] Optionally, and preferably transiently, disrupting the function ofthe dominant suppressor of gynoecium development or, preferablytransiently, reducing the expression of the dominant suppressor ofgynoecium development of a BC1 or BC2 or BCn (where BCn denotes highergeneration backcrossing for n generations) plant obtained in step [1] orstep [2] or step [3] to facilitate self-fertilization and select fromthe progeny thereof a plant that is homozygous for the ‘genetic trait’and represents a super-male

[5] Optionally obtaining doubled haploids of plants obtained in step [1]or step [2] or step [3] to select a plant that is homozygous for the‘genetic trait’ and represents a super-male.

[6] Optionally restoring the function or the expression of the dominantsuppressor of gynoecium development of a plant obtained in step [2] or[3] or [4] in such a way that the ‘hermaphrodite trait’ of said plant isno longer transferred to a next generation, thus becomes a supermale,which is preferably homozygous for the ‘genetic trait’.

The skilled person will appreciate that this step [6] will be necessarywhen in the pedigree of the plants obtained in [2], or [3] or [4] oreven [5], a permanent, rather than transient, loss of function orpermanent, rather than transient, reduced expression of the dominantsuppressor of gynoecium development was introduced, that may allow theunwanted transfer of the ‘hermaphrodite trait’ to a next generation andthus has to be restored into the male trait (thus at a common of atleast sufficient level of suppression of gynoecium development as innon-hermaphrodite or non-andromonoecious males).

The skilled person will appreciate that this method to apply a directcross of a first-degree relative and its super-male parent to obtainoffspring by said cross, hitherto has been impossible. For aconventional method of introducing a genetic trait into a super-male ahybrid can be made between a plant that has said ‘genetic trait’ bycrossing a first plant comprising said ‘genetic trait’ with a second‘super-male’ plant. However, the resulting hybrid will be male and cannever be directly crossed in a following generation to the super-malerecurrent parent. Instead it will take an additional cross of saidhybrid to a female plant first before the next hybrid, resulting fromthis latter cross, that retained the ‘genetic trait’ as female parentcan be crossed again to the super-male recurrent parent. In the methodprovided by the current invention a hybrid that has a first degreerelationship with a super-male can always be directly crossed with asuper-male in the following generation as this super-male or said hybridor both plant will be a ‘transgender male-to-hermaphrodite’ or a‘transgender male-to andromonoecous’ or a transgender ‘male- orandromonoecous-to-female’, which comprises the feminized trait.

An exception to the rule that a direct cross of a first-degree relativeand its super-male parent to obtain offspring by said cross will beimpossible could be provided when either the fist-degree relative or thesuper-male parent comprises ‘natural hermaphroditism or andromonoecy’.Such ‘natural hermaphroditism or andromonoeicy’ is not the result ofdisruption or reduced expression of the dominant suppressor of gynoeciumdevelopment but the result of naturally occurring unknown, non-Gynoeciumdevelopment suppressor GDS, ‘modifying genes’ such as have beenspeculated upon in the art as was illustrated in the literature outlinedin the previous paragraphs and thus differs from the ‘hermaphroditetrait’ as a tool to create syngeneic super-males provided by the presentinvention.

The manipulation of the GDS gene, which is responsible for expressingthe female suppresser, i.e. the suppressor of gynoecium development, canbe achieved in various manners. The GDS gene is represented by thehypothesized cDNA of the gene (SEQ ID NO:1):

ATGTCTGAAGCCTGGGTTTCTCGATTGACATCGGATATAGGGTGGCTCAATAGCACAAATGCCCTGATGGCGGAGGCCTGGAGTCGTCATTCAATCTACGACGTACCAGACACATTCAAAAGGATTAGCCCACAGATCCATAAGCCATCAACGTGCAGCATTGGACCACGGTACAATGGAGATCTGAATCTCCTTCGTATGGAACGTCATAAACACAGGGCGCTACTGAACTTCCTCATCCGATGTCAAGTGTCGATCCATGACATCATACGAGCCCTGAGGAAGAACCTGCACGATTTCAGAGCCTGCTATCAAGATCTTGACACCTTTTGGATGAAGAATGATGATGAGTTCCTAAAAATCATGATTTACGATGGGGCTTTCATGATTGAAATCATGATAGCGACCGTTGAACCATATGAGCGCACACCTTCTAGCTATCATGCCAAGGACCCAATATTCAAGAAGCCATACTTGGTCGAAGATCTTCGTGTAGATATGCTCAGGTTGGATAATCAAATTCCAATGAAGGTCCTGGAGATATTGTCTAAATTCTGCAAGAACAAGATCCAAAGCATTCATCAGCTGATCAGACATTTCTTCTTCCGCAAATATGAAGAGGGAAGATATGATATTAGCCAAACCTCTACGATATTTCACCTACCCGAGATAACAGGGCATCACCTACTGGATGTGTACAAAAAAACTCTTATACAGCATGGAGGTTATCATCACACCAGCAGTCGCCAACCACTATCGGCAGTTGAACTACAGGAGGCGGGCGTAATTTTCCAGTGCAGTGAAACGCTGTCATTGACAGATATATGCTTCACCAAAGGTGTCCTTTGCCTACCTGCAGTCGACGTTGACGAAGCATTTGAAGTTGTTATGCGGAATCTCATTGCCTATGAGCAAGCACATGGCGAAGGTCAAGAGGTAACATCCTATGTGTTTTTTATGGATGGCATTGTAAACAATGACAAAGATATTGCCTTGCTTCGAGAGAAGGGTATTATCAGGTCGGGGGTAAGCAGTGATAAGAGGATAGCCGATCTTTTTAATGGACTGACAAAAGGTATAGTTGCAAAAGTTGTCGACAATGTTGATGTTGATGTAACCAAGGACATCAATGAGTATTGCAATAGAAGATGGAACAGGTGGCAAGCCAACTTTAAGCAGAGATACTTTGCGAATCCATGGGCCTTTCCCGGGATTCATAAATGTTGATCTCAACGGTAGGGTTTCGTGCTGGGGTTTGAGTATCTGTGGAGCATTTAGTGTGAGAAAACTGTGCTTAATTTCGCTTCTCCACTATGAGAGTGGAGGAGCACAACTAATGGTATCCAGTGTAAATTTAACTCTTTGTTTGTGGCTTGAGAACAACATGTTCTTTATATAGCCTTTGACAATGTAATAGATAACATCAACTTCTTTGATACATACTAGCGAT ATTAGCATCCAAAAAAAAAA

This cDNA translates to the following protein (SEQ ID NO: 2):

MSEAWVSRLTSDIGWLNSTNALMAEAWSRHSIYDVPDTFKRISPQIHKPSTCSIGPRYNGDLNLLRMERHKHRALLNFLIRCQVSIHDIIRALRKNLHDFRACYQDLDTFWMKNDDEFLKIMIYDGAFMIEIMIATVEPYERTPSSYHAKDPIFKKPYLVEDLRVDMLRLDNQIPMKVLEILSKFCKNKIQSIHQLIRHFFFRKYEEGRYDISQTSTIFHLPEITGHHLLDVYKKTLIQHGGYHHTSSRQPLSAVELQEAGVIFQCSETLSLTDICFTKGVLCLPAVDVDEAFEVVMRNLIAYEQAHGEGQEVTSYVFFMDGIVNNDKDIALLREKGIIRSGVSSDKRIADLFNGLTKGIVAKVVDNVDVDVTKDINEYCNRRWNRWQANFKQRYFANPW AFPGIHKC

Alternatively, and depending on the way the genomic sequence is analyzedthe cDNA of the gene is represented by 5 other gene sequences, as listedin FIG. 3. These sequences are also identified in the presentapplication as “splice variants” or “splicing variants” or homologousAsparagus sequences (such as M4 and 3098, see Example 1) of SEQ IDNO: 1. It should further be indicated that these sequences are derivedfrom the genomic sequences that have been listed in FIG. 13

The term “GDS gene” as used in the present application is considered tocomprise all splice variants (including SEQID NO: 1) and all genomicsequences (including introns) that may be derived from the genomicsequences of FIG. 13, that encode a functional female suppressor orencode an homologous/orthologous gene. Any genetic constructs targetingthis gene or mRNA that is transcribed thereof are preferably targeted toexon 1, exon 2 (or exon 3) or the DUF247 domain. With respect to the DUFdomain, no exact consensus can be given. According to the EMBL-EBIdefinition of the DUF247 family, the domain is characterized by thefollowing database sequences, which are used as seed for building thefamily definition:

#=GS Q9SJR2_ARATH/47-434 AC Q9SJR2.1

#=GS Y3720_ARATH/48-447 AC Q9SD53.1

#=GS Q8L703_ARATH/63-464 AC Q8L703.1

#=GS Q9FK84_ARATH/46-474 AC Q9FK84.1

#=GS Q9FK85_ARATH/33-422 AC Q9FK85.1

#=GS Q01J11_ORYSA/116-543 AC Q01J11.1

#=GS Q9SNE9_ARATH/180-572 AC Q9SNE9.1

#=GS Q5XVA4_ARATH/115-523 AC Q5XVA4.1

#=GS Q9SN03_ARATH/92-493 AC Q9SN03.1

#=GS A0MF17_ARATH/106-485 AC A0MF17.1

#=GS A0MF16_ARATH/141-548 AC A0MF16.1

#=GS Q6ZC88_ORYSJ/184-584 AC Q6ZC88.1

#=GS Q0ISB3_ORYSJ/59-439 AC Q0ISB3.1

#=GS Q2QQW6_ORYSJ/36-452 AC Q2QQW6.1

#=GS Q2QQW3_ORYSJ/44-442 AC Q2QQW3.1

#=GS Q2R303_ORYSJ/44-473 AC Q2R303.1

#=GS Q1RU73_MEDTR/31-462 AC Q1RU73.1

#=GS Q6YPE9_ORYSJ/42-450 AC Q6YPE9.1

#=GS Q6YRM8_ORYSJ/34-376 AC Q6YRM8.1

#=GS 022159_ARATH/86-487 AC 022159.2

#=GS Q5S4X4_ARATH/111-507 AC Q5S4X4.1

#=GS Q6E287_ARATH/8-398 AC Q6E287.1

#=GS Q8VYN0_ARATH/16-440 AC Q8VYN0.1

#=GS Q1ZY19_BETVU/30-415 AC Q1ZY19.1

#=GS 049393_ARATH/295-669 AC 049393.2

#=GS Q9LFM8_ARATH/35-411 AC Q9LFM8.1

#=GS Q65XU3_ORYSJ/66-531 AC Q65XU3.1

#=GS Q65XU0_ORYSJ/49-551 AC Q65XU0.1

#=GS Q65XT8_ORYSJ/62-514 AC Q65XT8.1

#=GS Q9FP37_ORYSJ/53-496 AC Q9FP37.1

#=GS Q6ZKD8 ORYSJ/79-483 AC Q6ZKD8.1

#=GS Q69TN1_ORYSJ/150-572 AC Q69TN1.1

#=GS Q7XDW8_ORYSJ/117-510 AC Q7XDW8.1

#=GS Q0J689 ORYSJ/12-411 AC Q0J689.2

#=GS Q0J2S9_ORYSJ/46-452 AC Q0J2S9.1

#=GS Q0J2T1_ORYSJ/42-479 AC Q0J2T1.1

#=GS Q651E4_ORYSJ/52-471 AC Q651E4.1

#=GS Q2QPY1_ORYSJ/9-413 AC Q2QPY1.1

#=GS Q2QPX9 ORYSJ/148-562 AC Q2QPX9.1

#=GS Q656Q9 ORYSJ/57-451 AC Q656Q9.1

#=GS Q94D69_ORYSJ/72-478 AC Q94D69.1

#=GS Q94D66_ORYSJ/18-428 AC Q94D66.1

#=GS Q6ET10_ORYSJ/21-420 AC Q6ET10.1

#=GS Q8LJD1_ORYSJ/36-407 AC Q8LJD1.1

#=GS Q60E19_ORYSJ/30-431 AC Q60E19.1

#=GS Q10RD5_ORYSJ/49-462 AC Q10RD5.1

#=GS Q6H4T3_ORYSJ/102-533 AC Q6H4T3.1

#=GS Q6K301_ORYSJ/128-542 AC Q6K301.1

As has already be mentioned above, various mutants have been producedthat provide suppression of gynoecium development through a change inthe coding sequence or expression of the GDS gene. In a first embodimentthe interference with the female suppressor target gene consists ofpreventing transcription thereof. This can be achieved for instance bymeans of RNA oligonucleotides, DNA oligonucleotides or RNAi moleculesdirected against the target gene promoter.

Inhibition of the above mentioned gene expression is preferablyaccomplished by providing a plant with a construct which is able toexpress an inhibiting compound. Inhibition of gene expression refers tothe absence (or observable decrease) in the level of protein and/or mRNAproduct from the female suppressor target gene. Specificity ofinhibition refers to the ability to inhibit the female suppressor targetgene without manifest effects on other genes of the cell. Theconsequences of inhibition can be confirmed by examination of outwardproperties of the cell or the organism (in the specific case of theinvention, the sexual phenotype) or by biochemical techniques such asRNA solution hybridisation, nuclease protection, Northern hybridisation,reverse transcription, gene expression monitoring with a microarray,antibody binding, enzyme linked immunosorbent assay (ELISA), Westernblotting, radioimmunoassay (RIA), other immunoassays, and fluorescenceactivated cell analysis (FACS). Basically, four methods for inhibitionare known at this moment and included in this application: antisenseexpression, sense co-suppression, RNA-inhibition (RN Ai) and CRISPR-Casor CRISPR-Cpf mediated gene silencing. However, the invention is notlimited to these methods and any other method which causes silencing ofthe endogenous female suppressor gene is included.

For antisense expression, the nucleotide sequence of the femalesuppressor gene, or at least a part thereof of 19 nucleotides, usuallyat least 21-nucleotides or more: more preferably the GDS region, is putbehind a constitutive or sexual organ specific promoter in anti-sensedirection. After transcription of this nucleotide sequence an mRNA isproduced which is complementary to the mRNA formed through transcriptionof the endogenous female suppressor gene. It is well proven by now thatproduction of such an anti-sense mRNA is capable of inhibition of theendogenous expression of the gene for which it is complementary.Furthermore, it has been proven that to achieve this effect evensequences with a less than 100% homology are useful. Also antisensemRNA's which are shorter than the endogenous mRNA which they shouldinhibit can be used. Generally, it is accepted that mRNA sequences of 23nucleotides or more which have an identity of 70% or more will becapable of generating an inhibitory effect. The principal patentreference is EP 240,208 of Calgene Inc. There is no reason to doubt theoperability of antisense technology. It is well-established, usedroutinely in laboratories around the world and products in which it isused are on the market.

The second approach is commonly called sense co-suppression. Thisphenomenon occurs when the female suppressor gene or part of said geneis expressed in its sense direction. Although this kind of expressionwhen full length genes are used most often results in overexpression ofthe gene, it has been found that in some cases and especially in caseswhen a sequence shorter than the full length sequence is used,expression of this gene or fragment causes inhibition of the endogenousgene. The principal patent reference on sense co-suppression is EP465,572 in the name of DNA Plant Technology Inc.

Sense and antisense gene regulation is reviewed by Bird and Ray (Gen.Eng. Reviews 9: 207.221, 1991). Gene silencing can thus be obtained byinserting into the genome of a target organism an extra copy of thetarget female suppressor gene coding sequence which may comprise eitherthe whole or part or be a truncated sequence and may be in sense or inantisense orientation. Additionally, intron sequences which areobtainable from the genomic gene sequence may be used in theconstruction of suppression vectors. There have also been reports ofgene silencing being achieved within organisms of both the transgene andthe endogenous gene where the only sequence identity is within thepromoter regions.

The third possible way to silence genes is by using the so-called RNAitechnology, which covers all applications in which double-stranded RNAsare used to achieve silencing of an endogenous gene. As has beendemonstrated by Fire et al. (Nature, 391: 806.811, 1998) application ofa dsRNA of which one strand is at least partly complementary to theendogenously produced mRNA whether produced intracellularly or addedextracellularly is extremely capable of inhibiting translation of themRNA into a protein. It is believed that this phenomenon works throughthe intermediate production of short stretches of dsRNA (with a lengthof 23 nucleotides). To achieve production of dsRNA a construct is madeharbouring both a sense and an antisense nucleotide sequence (togetheralso called an inverted repeat) of at least 19, usually 23 nucleotidesor more, of which one is complementary to the endogenous gene whichneeds to he silenced. The sense and antisense nucleotide sequences canbe connected through a spacer nucleotide sequence of any length whichallows for a fold back of the formed RNA so that a double stranded RNAis formed by the sense and antisense sequence. The spacer then serves toform the hairpin loop connecting both sense and antisense sequence. Theorder of the sense and antisense sequence is not important. It is alsopossible to combine more than one sense-antisense combination in one andthe same construct. If the simple form is depicted as:prom—S—spac—AS—term, also the following constructs can be applied:prom—S1—spac—AS1—spac—S2—spac—AS2—term, orprom—S2—spac—S1—spac—AS1—spac—AS2—term. Variations in the built up ofthe construct are possible, as long as the end product of thetranscription of said constructs yields one or more dsRNAs.Alternatively, the double stranded structure may be formed by twoseparate constructs coding for complementary RNA strands, where RNAduplex formation occurs in the cell. In short notation these constructsthen look like: prom1-S1-term1 and prom2-AS1-term2. Prom1 and prom2 canbe the same or different but should both be constitutive orfruit-specific promoters, term 1 and term2 can be the same or different.Both constructs can be introduced into the cell on the same vector, butcan also be introduced using two different vectors.

RNA containing nucleotide sequences identical to a portion of the targetfemale suppressor gene are preferred for inhibition. RNA sequences withinsertions, deletions and single point mutations relative to the targetsequence have also been found effective for inhibition.

Thus, sequences with a sequence identity of less than 100% may be used.Sequence identity may be calculated by sequence comparison and alignmentalgorithms known in the art (see Gribskov and Devereux, SequenceAnalysis Primer, Stockton Press, 1991, and references cited therein),for instance by using the Smith-Waterman algorithm as implemented in theBESTFIT software program using default parameters (e.g. University ofWisconsin Computing Group). Thus, the duplex region of the RNA may bedefined functionally as a (double stranded) nucleotide sequence that iscapable of hybridising with a portion of the target gene transcript(e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. to 65° C.hybridization for 12-16 hours; followed by washing). The length of theidentical nucleotide sequences should be at least 23 nucleotides, butpreferably larger: 40, 50, 100, 200, 300 or 400 bases.

As disclosed herein, 100% sequence identity between the inhibitingconstruct and the target endogenous gene is not required to practice thepresent invention. Thus the invention has the advantage of being able totolerate sequence variations that might be expected due to geneticmutation, strain polymorphism or evolutionary divergence.

Thus also included in the invention are constructs having a nucleotidesequence under control of a sexual organ-specific promoter, wherein saidnucleotide sequence comprises a part of 19 r more nucleotides in a sensedirection, or in an antisense direction or in an inverted repeat form,of the sequence of SEQ ID NO:1 or sequences that: are more than 70%,preferably more than 80%, more than 90%, more than 95% or more than 98%identical therewith.

The recombinant DNA constructs for use in the methods according to thepresent invention may be constructed using recombinant DNA technologywell known to persons skilled in the art. The recombinant geneconstructs may be inserted into vectors, which may be commerciallyavailable, suitable for transforming into plants and suitable forexpression of the gene product in the transformed cells. Preferably usedare binary vectors which are useful for plant transformation usingAgrobacterium.

Alternatively, transcription is prevented by means of the expression ofa negatively acting transcription factor acting on the target genepromoter. Such negatively acting transcription factor can be natural orartificial. Artificial negatively acting transcription factors can beemployed by the overexpression of an engineered polydactyl zinc-fingertranscription factor coupled to a general transcription repressor.According to a further embodiment, the interfering with the target geneconsists of destabilizing the target gene mRNA, in particular’ by meansof nucleic acid molecules that are complementary to the target gene mRNAselected from the group consisting of antisense RNA, RNAi molecules.Virus Induced Gene Silencing (VIGS) molecules, co-suppressor molecules,RNA oligonucleotides or DNA oligonucleotides. In another embodiment theinterfering with the target gene consists of inhibiting the target geneexpression product. This can be achieved by means of the expressionproduct(s) of one or more dominant negative nucleic acid constructs,overexpression of one or more suppressors which interact with the targetgene product, or by means of one or more chemical compounds. A novel wayto introduce site-specific alterations in transcription of an(eukaryotic) gene is by a variation in the recently described CRISPR-Casgenetic engineering, homologous recombination system. (Cong L et al.Science 2013; 339: 819-823; Mali P et al. Science 2013; 339: 823-826:Cho S W et al. Nat Biotechnol 2013; 31: 230-232; Jinek M et al. Elife2013; 2: e00471). This variation entails the use of a Cas enzyme that isdefective in endonuclease activity, but which retains its ability, whenco-expressed with a gRNA, to specifically interfere with transcriptionalelongation, RNA polymerase binding or transcription factor binding. Thissystem is also indicated as CRISPRi. (Qi L S et al. Cell 2013; 152:1173-1183; Larson, M H et al 2013, Nature Protocols 8:2180.2196; Amelio,I. and Melino G., 2015, Cell Death & Differentiation; 22: 3.5)

The above-described systems are all systems that act on expression anddo not change the underlying genetic sequence of the gene. In thatrespect these systems are also relatively easy to switch on or switchoff at moments when suppression of expression is needed or whensuppression of expression is no longer needed. Such a switch can e.g.advantageously be effected by putting the expression of one or all ofthe components of the silencing system under control of a specific time-or location-restrained promoter. Such a promoter can be a promoter thatis only expressed during a particular stage of the development of theplant or in a specific organ of the pant. Examples for these arepromoters of genes that are specifically expressed during e.g. flowersetting or in plant reproductive organs. In another embodiment induciblepromoters may be used. Systems for introducing inducible expression inplants are commonly known (e.g. Borghi L 2010, Methods Mol Biol.655:65-75) In these systems addition of an exogenic factor, e.g. achemical compound such as alcohol or dexamethasone, may trigger start ordisruption of expression.

Next to changes in the expression of the gene, the gene itself may bechanged in such a way that no longer a functional protein is expressed.This may be achieved by mutating the gene. The one or more mutations canbe introduced randomly by means of one or more chemical compounds and/orphysical means and/or by insertion of genetic elements. Suitablechemical compounds are ethyl methanesulfonate, nitrosomethylurea,hydroxylamine, proflavine, N-methyl-N-nitrosoguanidine,N-ethyl-N-nitrosourea, N-methyl-N-nitro” nitrosoguanidine, diethylsulfate, ethylene imine, sodium azide, formaline, urethane, phenol andethylene oxide, Physical means that can be used comprise UV-irradiation,fast-neutron exposures X-rays and gamma irradiation. The genetic elementis a transposon, T-DNA, or retroviral element.

More efficient and targeted techniques are provided for by so-calledsite-directed mutagenesis techniques. Many systems for site-directedmutagenesis (SDM) are known to the skilled person, the most notoriousbeing nuclease based SDM systems such as zinc finger nucleases,transcription activator-like effector nucleases (TALENs), and LAGLIDADGhoming endonucleases (Curtin, S. J. et al., 2012, The Plant Genome5:42-50). Another technology for SDM is based on homologousrecombination with the target gene. The oldest is the Cre-Lox system,that has been extensively described. Already some time ago models havebeen presented by Bundock et al. (WO02/052026) and Prokopishyn et al.(WO03/062425). Very recently, the above discussed CRISPR-Cas system hasbeen proven very effective for SDM based on homologous recombination inplants (WO2014/144155).

As has been stated in the introduction, the skilled breeder (especiallyof dioecious plants, more particularly of asparagus) would also beinterested in enabling androecium development in plants. Enablingandroecium development in a female plant to essentially change thegender, would allow to obtain seeds from an originally female plant inthe absence of cross pollination (thus by self-fertilizaton) and wouldprovide the ability to obtain doubled haploids by in vitro androgenesisfrom such a plant. Androecium development or inhibition of androeciumdevelopment can be induced by modulating the expression of a geneproducing a dominant male stimulator.

This dominant male stimulator (also indicated as stimulator ofandroecium development or stimulator of anther development) is a proteinthat is encoded by a gene that is identical to or a homologue ororthologue of the TDF1 (defective in Tapetal Development and Function)gene, which is found in Arabidopsis AT3G28470 and in rice (osTDF1,LOC_Os03g18480). Preferably, the protein with this function, is encodedby the TDF1 ortholog from Asparagus officinalis as depicted in SEQ IDNO:5.

A functional homolog of the nucleic acid sequence is herein defined as anucleic acid sequence that has a high sequence identity with thesequence encoding the amino acid sequence depicted in SEQ ID NO: 5 andpreferably having an high sequence identity with the nucleotide sequenceof SEQ ID NO: 4 and which is expected to be able, when expressed in adioecious plant which does not bear anthers, to induce anther formation.

From a sequence comparison of these sequences with the sequences ofArabidopsis and rice it appears that the so-called R2 and R3 domains ofthe protein are the domains that provide the functionality that isneeded for the present invention. Thus, any protein sequence thatcomprises the R2 and R3 domains of the TDF1 gene and/or any nucleotidesequence encoding such a protein sequence, which sequence would befunctional when expressed in a dioecious plant is encompassed in thepresent invention. Especially preferred are sequences that comprise theR2 and R3 domains of the Asparagus officinalis TDF1 gene as depicted inSEQ ID NO: 5, which lie in the first 125 amino acids sequence of theprotein. Preferably said 112 and R3 domains are to be found from aboutaa 14—aa 57 (R2) and from about aa 70 to about aa 112 (R3)

Methods for using these nucleotide and/or amino acid sequences inbreeding of dioecious plants, preferably asparagus, have been discussedabove.

A further embodiment of the present invention is a method to detect ifthe plant has the property that is expected from the treatment. If thetreatment consisted in reducing the expression of the dominant gynoeciumdevelopment suppression gene, it, should be investigated whether theplant has become (more) feminized. This can be done by assessing thephenotype, i.e. waiting for the plant to be mature and checking whetherphenotypical characteristics of feminization appear. However, a fasterand more reliable method is using GDS marker assisted selection. In caseGDS marker assisted selection has identified a mutation that may causeloss of function of the GDS gene, the introduction of said mutation maybe guided by GDS marker assisted selection in further generations or theby molecular biological checks such as using markers that aresufficiently genetically linked to the mutation in the GDS gene,preferably at a genetic distance to the GDS gene of less than 50cM, morepreferably, less than 40cM, more preferably less than 30cM, morepreferably less than 20cM, more preferably less than 10cM, morepreferably less than 5cM, more preferably less than 2cM more preferablyless than 1cM to the M-locus to allow indirect. As will be establishedin the examples, the presence of one or two markers such as AO022,Asp1-T7, Asp2-Sp6, Asp4-Sp6, T35R54-1600seq, Asp80, Asp 432/448,Asp446,0 10A3_forward marker and 10B6_forward marker or CE64/CE66-HRMmay give away which genotype is present and hence which phenotype willresult from it. Next to the markers that are used in the experimentalpart of the present invention, it is also possible to use the geneticinformation of SEQ NO: 1 or SEQ NO: 3 to derive any markers or todevelop molecular based assays for determining the genetic make-up ofthe plant. Further, alternatively, markers may be derived from theM-locus_scaffold4 sequence, or Scaffold 905 which are presented in FIG.13.

In general, for selecting and crossing a plant in a method according tothe invention a marker is used to assist selection in at least oneselection step. It is known in the art that markers, indicative for acertain trait or condition, can be found in vivo and in vitro atdifferent biological levels. For example, markers can be found atpeptide level or at gene level. At gene level, a marker can be detectedat RNA level or DNA level. Preferably, in the present invention thepresence of such a marker is detected at DNA level, using the abovedescribed markers. Alternatively, a change in expression of the GDS genecan be assessed in plant parts by performing an immunoassay with anantibody that specifically binds the protein. Also primers such asdescribed in Table 3 hereinbelow, can be used to amplify the GDS gene,of which the presence can be tested by a probe that binds with thesequence of this gene, e.g. a sequence derived from SEQ ID NO: 1.Further, use can also be made of specific markers that are to be foundin the vicinity of the coding sequence, such as the markers that havebeen used in the experimental section of the present application. Incase of transgenic approaches selecting a transformed plant may beaccomplished by using a selectable marker or a reporter gene asdiscussed below.

In some cases it may be advisable to perform a method of the presentinvention through transient expression. Transient gene expression, as ise.g. achieved through agro-infiltration, is a fast, flexible andreproducible approach to high-level expression of useful proteins. Inplants, recombinant, strains of Agrobacterium tumefaciens can be usedfor transient expression of genes that have been inserted into the T-DNAregion of the bacterial Ti plasmid. A bacterial culture is infiltratedinto leaves, and upon T-DNA transfer, there is ectopic expression of thegene of interest in the plant cells. However, the utility of the systemis limited because the ectopic RNA expression ceases after 2-3 days. Itis shown that post-transcriptional gene silencing (PTGS) is a majorcause for this lack of efficiency. A system based on co-expression of aviral-encoded suppressor of gene silencing, the p19 protein of tomatobushy stunt virus (TBSV), prevents the onset of PTGS in the infiltratedtissues and allows high level of transient expression. Expression of arange of proteins was enhanced 50-fold or more in the presence of p19 sothat protein purification could be achieved from as little as 100 mg ofinfiltrated leaf material. Although it is clear that the use of p19 hasadvantages, an agro-infiltration without p19 can also be used to testthe functionality of candidate fragments and functional homologues, e.g.fragments and homologues that are used in RNAi constructs and/orCRISPR-Cas constructs.

In a particular embodiment of the invention it is preferred to restorethe disrupted or reduced expression of the dominant suppressor ofgynoecium development. Such a method could be provided by CRISPR-CAS ashas been shown for plants (Jiang et al., 2013) in which is wasdemonstrated that the disrupted GFP protein could be restored byCRISPR-Cas.

Further, the invention comprises a method to improve breeding indioecious plants comprising providing a plant in which the functionalexpression of the dominant male stimulator is restored and introducingsaid plant in inbreeding, backcross breeding, recurrent backcrossbreeding or double haploid breeding techniques. Such a restoration ofthe functional expression may be accomplished by complementation with afunctional copy of this dominant male stimulator.

In an alternative embodiment, the present invention comprises a methodfor self-fertilisation of dioecious plants wherein one or both of theparent plants is a plant in which the lack of functional expression ofthe dominant male stimulator is complemented by a functional copy ofsaid dominant male stimulator. When female plants are provided with afunctional copy of the male dominant stimulator, said plants will becomemore mascular and thus will produce anthers, and thus these plants maybe considered to be a hermaphrodite. As has been argued above, such ahermaphrodite plant may be used in several ways in the methods of theinvention.

Since a plant which is provided with a functional dominant malestimulator is producing anthers, the present invention is also directedto a method for n vitro androgenesis comprising providing a plant with agene that is able to produce such a functional protein. In order toproduce such a plant all methods for providing a plant or plant cellwith either a nucleic acid construct coding for the protein or theprotein itself can be used. Such methdos have been described brieflyabove and are well known to the person skilled in the art.

There are multiple ways in which a (recombinant) nucleic acid can hetransferred to a plant cell, for example Agrobacterium mediatedtransformation. However, besides by Agrobacterium infection, there areother means to effectively deliver of DNA to recipient plant cells whenone wishes to practice the invention. Suitable methods for deliveringDNA to plant cells are believed to include virtually any method by whichDNA can be introduced into a cell, such as by direct, delivery of DNAsuch as by PEG-mediated transformation of protoplasts, bydesiccation/inhibition-mediated DNA uptake (Potrykus et al., Mol. Gen.Genet., 199:183-188, 1985), by electroporation (U.S. Pat. No.5,384,253), by agitation with silicon carbide fibers (Kaeppler et al.,1990; U.S. Pat. Nos. 5,302,523; and 5,464,765), and by acceleration ofDNA coated particles (U.S. Pat. Nos. 5,550,318; 5,538,877; and5,538,880). Through the application of techniques such as these, cellsfrom virtually any plant species may be stably transformed, and thesecells may be developed further into transgenic plants.

In case Agrobacterium mediated transfer is used, it is preferred to usea substantially virulent Agrobacterium host cell such as A. tumefaciens,as exemplified by strain A281 or a strain derived thereof or anothervirulent strain available in the art. These Agrobacterium strains carrya DNA region originating from the virulence region of the Ti plasmidpTiBo542 containing the virB, virC and virG genes. The virulence (vir)gene products of A. tumefaciens coordinate the processing of the T-DNAand its transfer into plant cells. Vir gene expression is controlled byvirA and virG, whereby virA upon perception of an inducing signalactivates virG by phosphorylation. VirG, in turn, induces the expressionof virB,C,D,E. These genes code for proteins involved in the transfer ofDNA. The enhanced virulence of pTiBo542 is thought to be caused by ahypervirulent virG gene on this Ti plasmid (Chen et al. Mol. Gen. Genet230: 302-309, 1991).

After transfer of a nucleic acid into a plant or plant cell, it must bedetermined which plants or plant cells have been provided with saidnucleic acid. This may be done using molecular assaying techniques, suchas sequence alignment with molecular markers or PCR-based techniques,but it may also for example be accomplished by using a selectable markeror a reporter gene. Among the selective markers or selection genes thatare most widely used in plant transformation are the bacterial neomycinphosphotransferase genes (nptI, nptII and nptIII genes) conferringresistance to the selective agent, kanamycin, suggested in EP131623 andthe bacterial aphIV gene suggested in EP186425 conferring resistance tohygromycin. EP 275957 discloses the use of an acetyl transferase genefrom Streptomyces viridochromogenes that confers resistance to theherbicide phosphinothricin. Plant genes conferring relative resistanceto the herbicide glyphosate are suggested in EP218571. The resistance isbased on the expression of a gene encoding 5-enolshikimate-3-phosphatesynthase (EPSPS) that is relatively tolerant to N-phosphomethylglycine.Certain amino acids such as lysine, threonine, or the lysine derivativeamino ethyl cysteine (AEC) and tryptophan analogs like 5-methyltryptophan can also be used as selective agents due to their ability toinhibit cell growth when applied at high concentration. In thisselection system expression of the selectable marker gene results inoverproduction of amino acids by transgenic cells which permits thetransgenic to grow under selection. Suitable examples of reporter genesare beta-glucuronidase (GUS), beta-galactosidase, luciferase and greenfluorescent protein (GFP). However, preferably a marker-free approach,such as disclosed in WO 03/010319, is used, where the presence of theresistance gene(s) can be assayed with nucleotide sequence based assays.

Next to methods for introducing the (gene encoding for) the dominantmale stimulator, the expression of this protein may also be inhibited ashas been discussed above. Inhibition of gene expression or disruption ofthe gene may be accomplished using the techniques as identified abovefor inhibition of the dominant suppressor of gynoecium development. Asdiscussed above, inhibition of the dominant male stimulator, in additionto the inactivation of the female suppressor, should provide a femaleplant derived from a male or andromonoecious plants which is included asan example of a desirable feminized plant. Besides, inhibition of thedominant male stimulator might be useful in crosses where emasculationis required to provide an alternative for emasculation.

The invention is further illustrated in the following, non-limitingexamples.

Example 1 Genetic Analysis of Hermaphrodite Mutant 5375

Following anther culture of a heterozygous male (XY), Riccardi et al.(2010) obtained male (YY), female (XX) and the “5375” genotype, a rareexample of a completely hermaphroditic clone. This genotype for whichall flowers are hermaphrodite is distinct from andromonoecious genotypesthat have varying proportions of male and hermaphroditic flowers. Thecompletely hermaphroditic clone as mature plant showed the ability toproduce berries from all of its flowers, during vigorous growth in threesuccessive seasons which was a unprecedented high fruit set compared thefruit set of all male breeding stock ever evaluated by the institute ofCRA-ORL, Lodi, Italy. The hybrid plant that served as source materialused to obtain hermaphrodite 5375 was a male plant not capable ofproducing berries, and in case berries have been overlooked in someseasons it would only be few because source plants for anther culture,used in breeding preferably show very limited berry set.

In garden asparagus, two dominant alleles at two linked genes (A, F)have been hypothesized to control androecium development and repressionof gynoecium development, respectively (Bracale et al., 1990, Sex PlantReprod. 3:23.30; Bracale et al., 1991, Plant Sci 80:67-77).

In this model cited by Riccardi et al. (2010), females have the ‘aaff’genotype, heterozygous males have the ‘AaFf’ genotype, and super maleshave the ‘AAFF’ genotype. Riccardi et al (2010) speculated that arecombination event within the M locus of “5375” has produced a doubledhaploid and a totally hermaphroditic plant with an AAff genotype. Totest this hypothesis, several crosses were planned that were bothphenotyped with respect to flowering (to be classified as either female,male or hermaphrodite) and analyzed for sex linked markers. By using aset of highly variable single locus microsatellite markers it was laterdemonstrated that the particular hermaphrodite ‘5375’ was not a doubledhaploid but in fact is highly heterozygous. The assumption was made thathermaphrodite ‘5375’ represents a soma clone of a hybrid that donatedthe anthers used for tissue culture, rather than a doubled haploidoriginating from a pollen gamete in which a rare recombination tookplace. As a result the genotype ‘Aaff’ was considered to be moreappropriate. Under this model, this genotype retained its heterozygosityfor the dominant androecium development gene ‘Aa’ as observed in normalmales and it further carries two recessive alleles ‘ff’ of the dominantgynoecium development repressor gene because a loss of function mutationdisrupted the gynoecium development suppressor gene that was originallypresent in the heterozygous male that has donated the particular anther.In a scheme: AaFf (mutation)→Aaff

To test co-segregation of the hermaphrodite traits with the sexchromosome or ‘M-locus’, sex linked markers have been used. The firstmarker was a proprietary microsatellite marker, designated AO022, thatis derived from GenBank accession CV287860 which has the followingsequence (SEQ ID NO: 13):

GGCTCTTCTGGTTGGGATCAGTCATCGACTCAGCAAACTCAGCAAACTACTCCTGCAACTGGTTATGATTACTACAACCAGCAGCAGCAGCAGCAGCAGCAGCCACCAACATCAGCCCCAGCTGATAACACCAGCGCCTACAATTATTCCCAGCCTCATCCTGGTTATAGCTCTCAAGGTTCTTATACTGCTCAGCAGCCAACTTATGGTCAGGAAAACTATGCTGCTCCTGGTTATAACACTCAAACTCCCCAAACTGGTTATGATCAATCATACAATTCTGCACCTGCTTATGCTGGAGCTACCTCCACCAACCCCACTCAAGATGGATCTGCTGCATCCAATCAACCACCAAGCAGTGCTCCTGCTAGTTACCCCCCACAACCTGTGTACGGTGCACCTGCACCATTAACCCAACCCGGTTATGGACAGTCTCCTCAATCCCAGAAGCCACCGGCAACTCCGCCAGCTTATGCTCAAACAGGATATGGTACAAATACTGGATATGGTACACAGTACCAGCAGGTTCAGCCATATGGTGGGGGCCCACCAGCTGGCCAGGGAGGGTACGGTCAGCAGCAAGCATATGGTGATTCTTACGGCAGTGGTGGGTATTCCCAGCCACCGGCGTATGGGAGTGAGGGTGGTGCAGCTCCGGCGGCTCCTGGTGCAGTGACCAAGGCTTCTCCTCAGAGTTAGACGTGATGTATGGTAAGTTTTTGATGCGGTAGTTTTGCTTTAACACTTAGATTCCGGTAGAAGTTTAGATGTTGTAGTCTTGTGTTTTGCTCTGATTTGGTTTTGAATTTAGTAATGGTTTGTTAAGCTTTGTTGTTTCTGCGTGGGTGGAAATTCTGTATGTTTTCAAATTTGA

This marker has been previously tested in breeding and researchpopulations and always mapped at a genetic distance, varying from zeroto five centi-Morgan from the M-locus. The second marker was Asp1-T7published by Jamsari et al (2004), which has the sequence:

GAGTCGACCTGCGGGCATGCAAGCTTGGCGTGAATACGTTGCTGNGGATTCTCAATATGCGAGGCATTTGGAAGCACCAAAATCCGCACCCTACCGAGTACCCAAATCAAACACTTTCCATGGTGCCTTTCCACTATCTTCCTCACAATGTAATCTTCTAGTGAAATAAATGCAGTTACCTCTGTTGAGAGAGTGGATAGCCTTCTCATCAAAGAGCTAGCAGTGTTCACCTACCCCCGTGCTACAATGTTCACCTACCCCCTGCTACAGTGTTCACCTGTCCCAAATAGTGTTCACCTGCCCCCATGAGAAAATTTATAAATATCCCCCTAAGTTTGATTTGTAAGGTATCTCATTAGCAGAGAGAGAAAGAGAAAGATACAGATATAAGTGATATCATTGAGAGGTCTTGAGAGAGAGTTTGTAAGAATTCTTGGAGAGTATATTGAACAAGAGAGGGGGGTCTCTTTTATCTTTATTTTTGTACCTCGAAAGGGATA TAAAGGAATT

To find evidence for the hypothetical ‘Aaff’ genotype, several testcrosses were made. In the first pedigree, designated pedigree 1E,Hermaphrodite 5375 (Aaff) was allowed to self-fertilize. The resultingso called ‘S1’ or ‘F2’, progeny of pedigree 1E comprised bothhermaphrodites, females and no male plants. Flowers of this progeny wereanalyzed and fruit set (in insect free conditions) was recorded. Theobserved number of hermaphrodites and females was 166 and 56respectively, which follows a 3:1 ratio that is expected for a monogenicdominant gene conferring hermaphroditism. All hermaphrodites in theprogeny set berries under insect free conditions. It is important tonote that all flowers of stalks of mature hermaphrodite plants derivedfrom 5375 set fruit, thus produce berries, and that all berriescontained black colored fully developed seeds. Such well developed seedsare comparable to which are commonly observed in female plants Markeranalysis of the hermaphrodite parental plant 5375 disclosed its 161/169genotype for marker AO022, where 161 and 169 refer to estimated fragmentsizes in a capillary electrophoresis system. The estimated fragmentsizes may vary per capillary system but can be clearly distinguishedfrom each other by the skilled person. Further, the hermaphrodite 5375shows the presence of PCR marker Asp1-T7. It appears that the observedhermaphrodite trait is tightly linked to the AO022 microsatellite markerlocus. The AO022.169 allele is found in 163 out of 166 hermaphroditeplants, whereas this allele is lacking in 53 of 58 female plants. Allplants of this population were tested for the Asp 1-T7 male marker. All166 hermaphrodites tested had the Asp1-T7male marker allele, whereas all65 females tested lacked the Asp 1-T7 male marker allele. The resultsfor a subset of the pedigree 1E plants are summarized in Table 1a.

TABLE 1a Pedigree 1E. Result obtained for flower phenotypes and markersegregation of the progeny resulting from self-fertilization ofhermaphrodite 5375 that has a 161/169 genotype for marker AO022 andshows the Asp 1-T7 male diagnostic fragment when used as template DNAAO022 microsatellite genotype (161/169) self fertilized 161/169 169/169161/161 female 3 0 55 58 hermaphrodite 120 43 1 164 Asp1-T7- Asp1-T7-hermaphrodite (Asp1-T7 male allele male allele pres) self fertilizedpresent absent female 0 18 18 hermaphrodite 57 0 57

One can conclude that, with a few exceptions for the microsatellitemarker that must result from recombination events, plants that lack theAO022-169 allele (161/161 genotypes) and lack the dominant Asp1-T7 malemarker allele also lack anthers and thus are female. As a result it canbe speculated that the gynoecium suppressor has been lost inhermaphrodite 5375 (which then allows stigma development and fruit set)whereas the ability to produce anthers has been retained in heterozygouscondition that segregates in this cross and is linked to the geneticmarkers provided. In a second generation, twenty-six F3 familiesobtained from self-fertilization of 26 F2 plants that had the 161/169genotype for marker AO022 which is indicative of a Aaff genotype, werefurther phenotyped. Those family progenies varied in size between 4 and89 individuals per family. In all but three of these F3 families, againsegregation was observed for hermaphrodites and females for a totalnumber of 589 versus 193, respectively. This again is a 3:1 ratioexpected for a dominant gene conferring hermaphroditism where a dominantgene for androecium development segregates in a genetic background wherethe gynoecium development suppressor must be absent. In the other three(161/169-F2 plant derived) F3 families comprising eighty, twelve oreleven individuals only hermaphrodites were found thus no females. It islikely that for this particular plant a recombination event, hasoccurred between the microsatellite marker and the sex determinationgenes and that this particular self-fertilized plant, despite of its161/169 genotype had the AAff genotype. Of the largest family, tenplants were tested for the presence of the Asp 1-T7 fragment and indeednone of these plants lacked the male Asp1-T7 allele. Another fourteen F3families, varying in progeny size between 8 and 88, that were derivedfrom self-fertilized F2 plants which had the 169/169 genotype for markerAO022, indicative for a AAff genotype, produced a grand total of 324hermaphrodite siblings and no female plants. The results of the pedigree1E F3 crosses are shown in Table 2.

TABLE 2 Table showing the AO022 microsatellite alleles and Asp-1_T7marker results and plant phenotype of F2 families plus the segregationfor flower (and spontaneous berry set) phenotype as females (F) and/orhermaphrodites (H) in F3 families obtained for those individual F2plants. An ‘M’ indicated for marker AspT7-106 refers to the presence ofa PCR fragment diagnostic for the male specific region. Cross 1 (5375self = F2 plants) pseudo F2 plants) sample Pseudo F3 plants (plantAspT7- Phenotype n) 106 Phenot. AO022_1 AO022_2 F H 1 M H 161 169 4 8 5M H 161 169 5 13 8 M H 161 169 10 20 9 M H 161 169 18 69 13 M H 161 16913 76 24 M H 161 169 9 19 26 M H 161 169 3 3 36 M H 161 169 2 8 37 M H161 169 0 80 44 M H 161 169 23 68 53 M H 161 169 21 32 57 M H 161 169 513 66 M H 161 169 3 1 70 M H 161 169 3 12 75 M H 161 169 3 12 84 M H 161169 8 20 85 M H 161 169 9 43 87 M H 161 169 1 12 90 M H 161 169 28 62 95M H 161 169 4 12 99 M H 161 169 1 20 140 M H 161 169 7 23 141 M H 161169 0 11 169 M H 161 169 8 37 193 M H 161 169 5 6 200 M H 161 169 0 1234 M H 169 169 0 8 43 M H 169 169 0 13 7 M H 169 169 0 14 42 M H 169 1690 15 114 M H 169 169 0 16 96 M H 169 169 0 17 88 M H 169 169 0 21 4 M H169 169 0 22 6 M H 169 169 0 23 86 M H 169 169 0 24 71 M H 169 169 0 8861 M H 169 169 0 21 62 M H 169 169 0 21 82 M H 169 169 0 22

In a second pedigree designated pedigree 2E, the female double haploid‘5459’ was crossed to hermaphrodite ‘5375’. In the previously proposedgenetic model this is: 5495×5375=aaff×Aaff. For microsatellite markerAO022, plants 5495 and 5375, respectively showed the 166/166 and 161/169genotype. The progeny of this test cross 2E showed 64 hermaphrodites and83 female plants and no male plants. This does not differ significantlyfrom a 1:1 segregation ratio, which is consistent with a segregatingdominant gene for anther development. Further this population shows theentire absence of gynoecium development suppression that does notsegregate in this progeny which suggests, or at least does not reject,that 5375 effectively is homozygous for the loss of function of thegynoecium repressor gene. The segregation of phenotypic classes andmarkers are shown in Table 1b. The marker results show that, as alreadywas observed in cross 1E, that the AO022-169 allele is closely linked tothe hermaphrodite flower trait. The AO022.169 allele was present in 60out of 64 hermaphrodites whereas this allele is absent for 82 out of 83female plants. A subset of plants (11 hermaphrodites and 10 females)tested for Asp1 T7 show full linkage of the male allele and thehermaphrodite trait.

TABLE 1b Pedigree 2E. Result obtained for flower phenotype and markersegregation in the progeny of the cross: female 5459 × hermaphrodite5375 that respectively have the AO022 genotypes 166/166 and 161/169 andfor which only 5375 shows the diagnostic male Asp1-T7 fragment that isabsent in the female parent.: female (166/166) × hermaphrodite (161/169)166/169 161/166 female 1 82 83 hermaphrodite 60 4 64 female (Asp1T-7abs)) × Asp1-T7-male Asp1-T7-male hermaphrodite (Asp1-T7 pres) allelepresent allele absent female 0 10 10 hermaphrodite 11 0 11

In a third pedigree, designated pedigree 3E, the hermaphrodite plant‘5375’ was emasculated and crossed to double haploid super male ‘1770’.For this cross, twelve F1 plants were obtained. All of the twelve plantsof two expected different genotypes ‘AaFf’ and ‘AAFf’, were male andthus were incapable to produce fruits and seeds. This indicates that themale trait; the repression of gynoecium development is dominant over thehermaphrodite trait. It is thus established that the hermaphroditetrait; i.e. the ability of a plant, that has functional anthers, toproduce an androecium and fruits with seeds, is recessive. Evidence thatthe small number of plants indeed comprised both genotypes ‘AaFf’ and‘AAFf’, thus that indeed the ‘Af gamete’ (typical for the hermaphrodite)and not only the ‘af’ gamete (that can be obtained from common femalesand heterozygous males) contributed to the generation of this progenyfollows from marker analysis. Doubled haploid 1770 had a 166/166 markergenotype for marker AO022. The phenotypic results and the microsatellite marker results are shown in Table 1c. Seven F1 plants showedthe AO022 microsatellite marker 161/166 genotype. Because of the geneticlinkage between microsatellite AO022 allele ‘161’ and the femalephenotype (confirmed in pedigrees 1E and 2E) those plants, or at leastthe vast majority of those plants, must have resulted from a maternalgamete of hermaphrodite 5375 that has the female chromosome genotype‘af’ and a paternal gamete of doubled haploid super male 1770 that is‘AF’. As a result those plants are likely to have the ‘AaFf’ genotype.The remaining five plants had the AO022 microsatellite marker genotype169/166 and because of linkage between the microsatellite AO022 allele‘169’ and the hermaphrodite phenotype those plants, or at least the vastmajority thereof, must have resulted from a maternal gamete ofhermaphrodite 5375 carrying the male chromosome causing thehermaphrodite trait in alleles: ‘Af’ and a paternal ‘AF’ gamete ofdoubled haploid super male 1770. As a result those plants are likely tohave the ‘AAFf’ genotype. It is thus established that the hermaphroditetrait, i.e. the ability of a plant that has functional anthers toproduce an androecium and fruits with seeds, is recessive. The plantswere tested for marker Asp 1-T7 and all plants showed the male allele ofthis male specific marker.

TABLE 1c Pedigree 3E. Result obtained for flower phenotypes and markersegregation for F1 cross 5375 × (5375 × 1770) in which the F1 plant withgenotype 169/166 was used to pollinate emasculated 5375: 5375 (161/169)× 1770 (169/166) 169/166 161/166 hermaphrodite 0 0 0 male 5 7 12

TABLE 1d Pedigree 3E. Result obtained for flower phenotypes and markersegregation of pseudo test cross 1800 × selected F1 (5375 × 1770) whichfor their markers corresponds to 1800 (166/166) × selected F1 (169/166):1800 (166/166) × selected F1 (169/166) 169/166 166/166 hermaphrodite 111 12 male 0 12 12

TABLE 1e Pedigree 3E. Result obtained for flower phenotypes and markersegregation of pseudo test cross 1800 × selected F1 (5375 × 1770) whichfor their markers corresponds to1800 (HRM curve ‘T deletion’) × selectedF1 (HRM melting curve WT): 1800 ( ) × selected F1 DUF247 T DUF247 WT (Tdeletion/WT sequence) deletion sequence hermaphrodite 12 0 12 male 0 1212

In a next generation for pedigree 3E, a pseudo test cross was made. Asingle male plant derived from the cross 5375×1770 was selected for itsAO022 marker genotype 169/166 that, because of linkage between themarker and sex determination genes, almost certainly will have thegenotype AAFf. This selected plant was crossed to a doubled haploidfemale plant ‘1800’ that had the 166/166 genotype for marker AO022. Informula: 1800× selected F1(5375×1770)=166/166×169/166=aaff×AAFf. Thisfamily segregated for 121 males versus 118 hermaphrodites consistentwith a 1:1 ratio expected for the segregation of the dominant gynoeciumdevelopment suppressor Ff versus ff. It is also consistent with thegenetic model that predicts that all siblings will be heterozygous ‘Aa’for the androecium development gene and thus all have anthers. For asubset of twenty-four plants of this pseudo testcross, the AO022microsatellite genotypes was determined. The results are shown in Table1d. Eleven of twelve hermaphrodites had a 166/169 genotype. Onehermaphrodite, a possible recombinant between the 166 allele and thegynoecium development repressor gene, had a 166/166 genotype. All twelvemale plants had a 166/166 genotype. This confirms linkage between theAO022 microsatellite paternal marker allele ‘166’ (originating from themale grandfather 1770) and the gynoecium development repressor. Itfurther indicates that the absence of the gynoecium developmentrepressor allele, linked to the AO022 169 paternal allele of thehermaphrodite grandparent, allows gynoecium development.

All of the above crosses teach that the segregation observed isconsistent with an ‘Aaff’ genotype for hermaphrodite clone 5375 that isheterozygous for the androecium development gene as in common males butlacks a functional allele of the gynoecium development repressor gene.The recessively inherited hermaphroditism is linked to the sexchromosome of asparagus and the genetic analysis presented here,provides for a method in which the genetic linkage between the trait andmarkers on the sex chromosome can be tested or verified. It is obviousto the skilled person that other markers can be used for testing thelinkage of the hermaphrodite trait to the sex chromosome as well.

The results of the crosses further teach that the hermaphrodite is ableto self-fertilize and provides offspring in further generations, whichcauses inbreeding. Inbreeding in this example can be inferred formanalysis of the AO022 genotypes. For instance, for pedigree 1E theheterozygosity at the AO022 locus is reduced by 50%. It is conceivableby any geneticist or breeder that this kind of inbreeding can reduceheterozygosity at any other locus. Further it will be clear to theskilled person that compared to full-sib mating the inbreeding byself-fertilization which is presented here occurs more efficiently. Infull-sib mating, like the subsequent crossing of sisters and brothers,it takes three times more generations to achieve a similar decrease inheterozygosity compared to self-fertilization (Bos, 1985. Thevenin,1967; p108). It is further conceivable that one can easily get rid ofthe hermaphrodite trait in a particular inbreeding generation byselecting against the androecium development gene (e.g. by using linkedmarkers) to finally obtain inbred female plants that will not pass thehermaphrodite trait to a next generation in case this is no longerdesired (e.g. in a commercial F1 hybrid). Therefore, a method isprovided to obtain inbred lines by self-fertilization that does notdepend on autosomal modifiers enabling self-fertilization (for thosemodifiers see Franken, 1969, 1970). Instead, the inbreeding in thepresent invention relies on the selection for a recessive allele thatallows gynoecium development linked to a dominant gene ‘Af’ that allowsandroecium development followed by later selection against this alleliccombination ‘Af’ of two linked genes to obtain common female inbredlines.

Because there is co-segregation of the hermaphrodite trait and markerAsp1-T7 that is indicative for a chromosome segment unique to maleplants, the previously proposed theory of Riccardi et al (2010) that arecombination event has ‘replaced’ the gynoecium repressor located on amale specific chromosome segment for a female chromosome segment thatnaturally lacks a gynoecium repressor was rejected. Instead, it washypothesized that a mutation has taken place in the gynoecium repressoron the male chromosome segment that is still present in thehermaphrodite plant and has not been lost by recombination. Thismutation can be transmitted to a next generation and shows Mendeliansingle locus segregation.

As a result efforts were aimed at finding a gene in which a mutation hasoccurred.

The laboratory of Dr. James H Leebens-Mack (University of Georgia atAthens, USA) has worked on a draft genome sequence of doubled haploidsuper male DH00/086 (version 1.0) in collaboration with the BeijingGenomics Institute at Shenzhen, China (BGI).

For this, genomic DNA (gDNA) isolated from spear or fern tissue ofDH00/086 was isolated and pooled for Illumina HiSeq sequencing. Briefly,the pooled gDNA was prepared for shot gun library preparation by strictfragmentation and end repair of gDNA, adapter ligation, size selection,PCR amplification, library purification and Quality Control. A total of9 short-insert paired end libraries and 6 long-insert paired endlibraries (Mate pair) were used for Next Generation Sequencing (NGS); 21flow channels were prepared and the libraries were sequenced inHiseq2500 2×100nt, paired-end mode. The data was collected and filteredaccording to Quality scores in Illumina pipeline 1.8. A total of 163Gigabase of sequence passed the Quality criteria corresponding toapproximately 123× coverage of the haploid genome of Asparagusofficinalis. The De Novo assembly of was conducted in the SOAPdenov2pipeline with a multiple k-mer strategy (Luo et al., 2012, Peng et al,2012). SOAPdenovo2, as with SOAPdenovo, is made up of six modules thathandle read error correction, de Bruijn graph (DBG) construction, contigassembly, paired-end reads mapping, scaffold construction, and gapclosure. The de novo assembly has 24,113 scaffolds with prefix ScafSeq-,of which 115 were pseudoscaffolds made up of alignment of genomicsequences which most likely map to the M-locus and surrounding regions.The pseudoscaffolds have the prefix M-locus_scaffold-. The genomicsequences used for alignment included Bacterial Artificial Chromosome(BAC) contig sequences derived from two BAC-libraries constructed fromHigh Molecular Weight genomic DNA of the genotypes DH00/086 (supermale)and DH00/94 (female) (Leebens-Mack, J H, personal communication 2010).The libraries were screened with molecular markers genetically coupledto M-locus phenotypes and the BAC DNA of candidate clones wassubsequently sequenced using Illumina TruSeq cluster chemistry for theGenome Analyzer Hx system. One useful statistic of De Novo assembliessuch as the 24,113 scaffolds-containing assembly of Asparagusofficinalis is the N50 value. Briefly, contig or scaffold N50 is amedian statistic such that 50% of the entire assembly is contained incontigs or scaffolds equal or larger than this value. The resulting ofassembly of the data of Asparagus officinalis exhibited a contig N50 of21,179 and scaffold N50 of 301,040 representing 80% of its haploidgenome. The consensus sequences of the scaffolds were used forannotation purposes such as putative repetitive elements and ab initiogene prediction and served as Reference Genome in both cDNA read mappingexperiments, referred to as RNA-Seq experiments and genomicre-sequencing experiments of several genotypes of Asparagus officinalis.The Reference Genome used is referred to as Asparagus Genome ScaffoldV1.10 (AGS V1.10) the annotation metadata were stored as individualfiles in AGS V1.10 based relational databases.

A number of methods were used to screen AGS V1.10 for all known classesof repetitive elements as well as newly found predictions of repetitiveelements including plant transposon elements. LTRharvest is a softwarepackage that computes boundary positions of Long Terminal Repeatretrotransposons in genomic sequences (Ellinghaus et al., 2008).LTRharvest was used in default and manually set similarity indices andoutput files included predictions in FASTA format and GFF3 format.Repeat Explorer is a python script software suite that includesutilities for characterization of repetitive sequences and transposableelement coding sequences in NGS data (Novak et al 2010). Next to thecommand line versions RepeatExplorer is accessible on a Galaxy-based webserver: www.repeatexplorer.org (Novak et al., 2013). RepeatMasker is aprogram that screens DNA sequences for interspersed repeats and lowcomplexity sequences. RepeatMasker (Institute for Systems Biology,Seattle, Wash.) is a set of BLAST-based programs that aligns input querysequences to curated databases of repetitive elements and output filesinclude a masked query sequence in which the nucleotides of predictedrepetitive elements are replaced by the symbol N. The query AGS V1.10was masked using RM13last at default sensitivity. The masked output file(nnAGS V1.10) was used for ab initio gene prediction. Basically, theprograms use trained sets of algorithms to collect evidence for genes byidentifying candidate signal sites such as promoter, translationalstart, termination, splice donor, splice branch and splice acceptorsites, suggested by given sources of gene evidence. Ab initio geneprediction was performed using BGI pipelines (Fgenesh and GlimmerHMM) indefault settings resulting in combined GLEAN files for gene evidence(Elsik, 2007). The set comprised 28,288 predicted protein-coding geneswith an average CDS length of 1006 bp and on average 4.75 exons perpredicted transcript. In addition, the SNAP (Semi-HMM-based Nucleic AcidParser, Korf, 2004) software package with Viridiplantae settings wasused to predict gene models. A total of 24,116 genes were predicted bythe SNAP algorithms.

RNAseq

Two RNA-Seq experiments were performed. The first experiment wasdesigned to identify differentially expressed transcripts betweenfemale, male, and supermale Asparagus genotypes and subsequently mapthese transcripts to the AGSV1.10 genome assembly. In total, 13 Limgroupasparagus lines, namely 9Female (9F), 9Male (9M), 88F, 88M, 88superMale(88supM). 89F, 89M, 89supM, 103F, 103M, 103supM and the male DH linesDH00/86 and DN3389 were processed (Limgroup BV, Horst, The Netherlands).Briefly, total RNA was isolated from flower buds using RNeasy Plant MiniKit protocols (Qiagen GmbH, Hilden Germany) and RNA quality was assessedwith Agilent RNA Bioanalyzer protocols (Agilent, Santa Clara, Calif.).The RNA was converted into double-stranded cDNA and prepared forIllumina NGS shot gun library preparation by adapter ligation, sizeselection, PCR amplification, library purification and Quality Control.A total of 13 short-insert paired end libraries were used for NGS; 3flow channels were prepared and the libraries were sequenced inHiseq2500 2×100nt paired-end mode. The data was collected and filteredaccording to Quality scores in Illumina pipeline 1.8. A total of 500Million reads passed the Quality criteria. De novo transcriptomeassembly was conducted in the Trinity software package (Grabher et al.,2013). Trinity combines three independent software modules: Inchworm,Chrysalis, and Butterfly, applied sequentially to process large volumesof RNA-seq reads. Trinity partitions the sequence data into manyindividual De Bruijn Graphs, each representing the transcriptionalcomplexity at a given gene or locus, and then processes each graphindependently to extract full-length splicing isoforms and separatetranscripts derived from paralogous genes. After normalization of thepaired end reads, 276,556 sequences were assembled with a total lengthof 378 Mb and N50 of 2386. The 13 paired end read data sets were mappedback on the De novo assembly and data for the genotypes was compared tocall for gender specific expressed Single Nucleotide Polymorphisms(eSNPs) and short insertion/deletions (indels) using the softwarepackage vcftools (variant call format, Wellcome Trust Sanger Institue,Cambridge, UK). A number of stringency settings was performed andreviewed. It was concluded that no strict gender specific eSNPs orindels could be called for further validation. The RNA-Seq data was alsoused to address differential expression of genes in the aforementioned11 LimGroup samples using the Cufflinks software package versionCufflinks 2.2.1 (Trapnell et al., 2010).

Cufflinks assembles transcripts, estimates their abundances, and testsfor differential expression and regulation in RNA-Seq samples. Itaccepts aligned RNA-Seq reads and assembles the alignments into a set oftranscripts. Cufflinks then estimates the relative abundances of thesetranscripts based on how many reads support each one. Briefly, theRNA-Seq data was aligned to the Reference AGS V1.10 using TopHat 2.0.13(Kim et al., 2013) with default stringency settings. TopHat aligned theRNA Seq reads to the rmAGS V1.10 Reference and analyzes the mappingresults to identify splice junctions between the exons. The data wasprocessed in Cufflinks using the Cuffdif2 algorithm (Trapnell et al.,2012) to identify and quantify differentially expressed transcripts.Comparison of the expression revealed a pattern both between the linesand the genders in general. Cluster analysis of expression patternsshowed that three clusters appear, related clusters for the 88 and 89genotypes and a third cluster having the 9 and 103 genotypes expressionpatterns. The comparison of Male versus Female expression for allgenotypes shows that 269 genes were significantly upregulated in theMale samples and 2 downregulated. The comparison of Supermale versusFemale expression for all genotypes shows that 434 genes wereupregulated and 49 downregulated. A number of genes involved in antherdevelopment were found to he differentially expressed in Supermalesversus Females including the genes orthologous to genes for ABORTEDMICROSPORES AMS' and MALE STERILITY MS2 annotated in Arabidopsisthaliana. A list of at least 40 genes showed no expression in Femalesamples.

The second RNA-Seq experiment was designed to study whole genome geneexpression in flower buds obtained from different genotypes of Asparagusof particular developmental stages. The genotypes and their relatedsamples selected for RNASeq analysis were the following: DH Male1770=sample 1; DH Female 1800=sample 2; Herma 5375=sample 3; plants AAffHerma of Pedigree 1E=Bulk 1 and 4 plants AaFf Males of pedigree 3E=Bulk2. From each plant, three flower button stages were sampled: A)pre-meiosis (1.0-1.2 mm long for Herma and Male, 0.8-1.0 mm for Female);B) uni-nucleated microspores (1.6-1.8 mm), or just developed ovary(1.2-1.4 mm); C) fully developed carpels (just before sepal opening).Briefly, total RNA was isolated from flower buds using a NucleoSpin RNAPlant Kit (Macherey-Nagel GmbH & Co. Duren, Germany)) and RNA qualitywas assessed with Agilent RNA Bioanalyzer protocols (Agilent, SantaClara, Calif.). The RNA was converted into double-stranded cDNA andprepared for Illumina NGS shot gun library preparation by adapterligation, size selection, PCR amplification, library purification andQuality Control. A total of 13 short-insert paired end libraries wereused for NGS; 2 flow channels were prepaired and the libraries weresequenced in Hiseq1000 2×100nt paired-end mode. The data was collectedand filtered according to Quality scores in Illumina pipeline 1.7. TheRNA-Seq data was aligned to the Reference AGS V1.10 using TopHat 2.0.13(Kim et al., 2014) with sensitive stringency settings(—b2-very-sensitive) and a large maximum intron size (40 kb). TopHatannotation data were stored as metadata to AGS V1.10 and loaded asindividual tracks in the Integrated Genomics Viewer (IGV, Robinson etal., 2011). In IGV, genomic scaffolds of AGS V1.10 can be inspectedindividually.

The laboratory of Dr. Leebens-Mack also applied AUGUSTUS Gene Prediction(Hoff et al., 2013) and EVM (Evidence Modeler, Haas et al., 2008) toaggregate gene model predictions from multiple sources. AUGUSTUS geneprediction involves two subsequent steps: creating a training set forAsparagus and the actual gene prediction. The training softwareautomatically generates gene sets from genomic sequences and the set ofTrinity assemblies and subsequently trains AUGUSTUS parameters for a newspecies. These new parameters and the supplied extrinsic evidence areapplied in the gene prediction modules. EVM was used to integrate allgDNA and RNA-Seq data available. The software combines ab initio genepredictions and transcript alignments into weighted consensus genestructures. For Asparagus, this included the GLEAN, SNAP, Trinity,Cufflinks and AUGUSTUS data sets. The highest weight was given to theCufflinks data and the lowest weight to the GLEAN data. A total of24kGene Models was annotated. The gene prediction metadata were storedas individual files in AGS V1.10 based relational databases.

Re-sequencing includes mapping or alignment of reads to the Referenceand error correction. For this, short-insert paired end Illumina HiSeqsequencing data (BGI, Shenzhen, China Shenzhen, China Shenzhen, China)were obtained of the Asparagus officinalis genotype DH00/094. DH00/094is a female doubled haploid obtained by tissue culture from the samehybrid from which DH00/086 originates. The data included 100nt pairedend reads representing approximately 40× genomic coverage. The reads ofboth DH00/086 and DH00/094 were aligned to the Reference genome usingthe Burrows-Wheeler Aligner in the software package bwa-MEM with defaultsettings (Li and Durban, 2009) as well as the more recently developedultrafast short-read aligner included in the software package Bowtie2(Langmead et al., 2012). The DH00/094 mapping was used to call forgender specific SNPs and short indels using the software packagevcftools (variant call format, Wellcome Trust Sanger Institue,Cambridge, UK). A number of stringency settings was performed andreviewed. Initially, SNPs were found in at least 3,195 gene Models. There-sequencing metadata were stored as individual files in AGS V1.10based relational databases. All metadata to AGS V1.10 including theaforementioned LTR-harvest data, gene predictions, Trinity RNA-Seqassemblies, Cufflinks annotations and re-sequencing data were stored asindividual tracks in the genome browser JBrowse 1.11.4 (Generic ModelOrganism Database project GMOD, 2013). Tracks can be vizualized for allgenomic scaffolds of AGS V1.10 individually.

Markers

All available genetic and molecular data of genomic sequences known tobe related to the M-locus in Asparagus officinalis were used as querysequences in local alignment searches (BLAT, BLAST, Althschul et al.,1990) in a blast database of the reference genome scaffolds AGS V1.10.The searches were performed in default settings. These molecularsequences included published genetic markers closely linked to theM-locus designated Asp1-T7, Asp2-Sp6, Asp4-Sp6 (Jamsari et al., 2004),T35R54.1600seq (Kanno et al., 2013) and genetic markers developed byLimgroup designated Asp80, Asp432/448, Asp446, 10A3_forward marker and10B6_forward marker. Asp1-T7 (510 nt) has 98.37% Identity to scaffold905at position 305206-304717 and related pseudomolecule M-locus_scaffold4(ML4) at position 5470-5959. Asp2-Sp6 (634nt) has 98.85% Identity toscaffold905 at position 307405-306883 and ML4 at position 3271-3793 and96% Identity to scaffold199 at position 464878-464359. Asp4-Sp6 (443 nt)has 96.62% Identity to scaffold997 at position 224027-224469 and showshigh Identities (>80%) to a further 303 genomic scaffolds. The sequenceof Asp-Sp6 was annotated as LTR-retrotransposon, subclass Ty1-copiarelated. The sequence T35R54 (1586 nt) is part of a highly repetitiveregion in the genome of Asparagus and has 100% identity to 25 genomicscaffolds, among which ML4 at position 22173-21039. Asp80 aligns toscaffold1194, Asp432/448 to scaffold206 and Asp446 to scaffold 1539. Thesequences of 10A3_forward marker and 10B6_forward marker align with 100%Identity to scaffold997 and related pseudomolecule M-locus_scaffold2.Since three of closely linked sequences align to a small region inscaffold905 and ML4, these scaffolds were prioritized as subjects tofurther study. EVM data show fifteen (15) Gene Models in scaffold905(351847 bp) and three (3) Gene models in ML4 (94405 bp). Two (2) EVMannotations are in close vicinity to the positions of the markersequences Asp 1-T7, Asp2-Sp6 and T35R54: evm_1.TU.M-locus_scaffold4.1(type: mRNA, 189 bp) and EVM_1 prediction M-locus_scaffold4.2 (Type:Gene 2640 bp).

Both EVM annotations were translated and used as query in the alignmentsoftware BLASTP using a database of the non-redundant protein sequences(nr) of Genbank CDS translations plus protein sequences in the databasesPDB, Swissprot, PIR and PRF (ncbi.nlm.org updated 2015 Jan. 5, 54183042sequences). The sequences were limited to the Viridiplantae [ORGN]including a filter for low complexities. All other settings weredefault. The translation of evm_1.TU.M-locus_scaffold4.1 has nosignificant hits in the database. The translation of EVM_1 predictionM-locus_scaffold4.2 has a highly significant Identity (38.54%) to boththe hypothetical protein VITISV_031339 of Vitis vinifera (Hit: CAN82114,Id: 147844299) and the predicted UPF0481 protein AT3G47200-like of Vitisvinifera (Hit: XP_010657662, Id: 731377489). Both entries were used inthe conserved domain alignment at NCB′(http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) and have highsimilarity to members of the family Pfam03140, Plant protein of unknownfunction (Domain of Unknown Function, DUF247). The Pfam database is alarge collection of protein families, each represented by multiplesequence alignments and hidden Markov models (HMMs). Current version isPfam 27.0 (March 2013, 14831 families). The family Pfam03140 (PF3140)consists of 48 members and belongs to DUF247 Superfamily cI03911. Thefunction of the plant proteins constituting this Superfamily is unknown.The DUF247-like gene sequence, which was temporarily called‘DUF247-like’ was used as Query in a database of the Reference genomescaffolds AGS V1.10. Next to scaffold905 and pseudomolecule ML4 twoDUF247-like sequences from two unrelated scaffolds were returned: regionDUF247-like scaffold 3098 (1965 bp) and the region DUF247-likescaffold10515 (1422 bp). An alignment was created with Clustal Omega(Sievers et al., 2011) in standard settings. DUF247-like scaffold10515aligns with 91% Identity from position 69 to 1186 in CDS2 and DUF-likescaffold3098 aligns with 92% Identity to position 1 to position 1970 inthe second intron. The alignment is shown in FIG. 2. The DUF-likescaffold3098 gene is predicted in EVM1 and AUGUSTUS annotations(scaffold3098 scaffold3098:95411.97134 (+strand) class=gene length=1724)and supported by Cufflinks annotations (TCONS_00149163). The sequencesof the above-discussed scaffolds can be found in FIG. 13.

In The Arabidopsis Information resource (TAIR10) the query termAT3G47200 was used and returned 2 loci matches, AT3G47200 and AT3G47210with 5 distinct gene models. It was decided to investigate relationshipsof all Arabidopsis gene models found by BlastP in TAIR Protein(proteins) sequences using the translation of EVM_1 predictionM-locus_scaffold4.2 as query. The highest scores were AT3G50150.1, Plantprotein of unknown function (DUF247), chr3:18595809-18597551 REVERSELENGTH=509 (Score=201 bits (511), Expect=8e-52, Identities=133/425(31%), Positives=216/425 (50%), Gaps=34/425 (8%)) and AT3G50160.1, Plantprotein of unknown function (DUF247), chr3:18598826-18600903 REVERSELENGTH=503 (197 bits (502), Expect=8e-51, Identities=132/413 (31%),Positives=207/413 (50%), Gaps=29/413 (7%)). Notably, the AT3G47200.1gene model shows less identity: (DUF247), chr3:17377658-17379088 REVERSELENGTH=476 (Score=130 bits (326), Expect=2e-30, Identities=104/417(24%), Positives=195/417 (46%), Gaps=52/417 (12%)). The Arabidopsisgenes with significant identities are listed in table X, column AGICode. The translation of the paralogous sequences of DUF-likescaffold3098 also returns the highest scores for AT3G50150.1, Plantprotein of unknown function (DUF247) aligning a less significantfraction of 181/454 amino acids. TAIR description of AT3G50150 andATG3G50160 contains: Plant protein of unknown function (DUF247);INVOLVED IN: biological_process unknown LOCATED IN: plasma membrane:EXPRESSED IN: inflorescence meristem, petal; hypocotyl, root: EXPRESSEDDURING 4 anthesis.

The external links were accessed for more information. The PlantProteome database (PPDB) returns another four (4) gene models forArabidopsis and 10 gene models in Oryza sativa datasets. The SubCellularProteomic Database (SUBA3) houses large scale proteomic and GFPlocalization sets from cellular compartments of Arabidopsis. It alsocontains precompiled bioinformatics predictions for protein subcellularlocalizations. A new dataset of protein-protein interactions hasrecently been added. The predicted subcellular location for theAT3G47200 protein (nucleotide sequence derivable from GenBank accessionno. AK221225.1) from both annotations as well as Ms/Ms experimentspoints to the plasma membrane, the peroxisome and plastid. None of theother databases has relevant information stored for this protein withone exception: the Phytozome Plant Gene Families databases(www.phytozome.net) displays cluster 38694300 having 922 members across40 genome sequences of representatives of the Glade of Viridiplantae,including green algae. The ontologies associated with this familyinclude PF03140 (DUF247) and Biological_process GO:0008150; when thisterm is used for annotation, it indicates that no information wasavailable regarding the biological process of the gene productannotated. The evidence code ND, no data, is used to indicate this. Asmall number of ontologies include PF00043, the PfamA annotation ofGlutathione S-transferase, C-terminal domain. Next to detoxificationfunctions in eukaryotes, the domain is also found in proteins whichharbor no such activities, such as the HSP26 family of stress-relatedproteins, which include auxin-regulated proteins in plants. Toinvestigate expression profiles of family members of PF03140 inArabidopsis, the GENEVESTIGATOR interface was explored(www.genevestigator.org, NEBION AG, Zurich, Switzerland). GENEVESTIGATORis a high performance search engine for gene expression analysis. Itintegrates thousands of manually curated, well described publicmicroarray and RNAseq experiments and visualizes gene expression acrossdifferent biological contexts such as tissues or genotypes. In the PlantBiology database 10 species are described including 10,773 samplesrepresenting 600 studies of Arabidopsis thaliana microarray experiments.For these studies, the GeneChip® Arabidopsis ATH1 Genome Array(Affymetrix, Santa Clara, Calif.) experiments are curated. ATH1 isdesigned in collaboration with the former TIGR institute (now CraigVenter Institute, Rockville, Mass.) and contains more than 22 500 probesets representing approximately 24,000 genes. In the CONDITIONSenvironment of GENEVESTIGATOR all ATH experiments were selected toinvestigate gene expression of the genes in table X, includingAt2G38540, the ATH TDF1 gene model for DEFECTIVE in TAPETAL DEVELOPMENT,as control (Jun Zhu, 2008). AT3G47200 is not found in the GENEVESTIGATORATIII data. An overview of the expression levels using all ATHexperiments in 10 developmental stages of Arabidopsis displays arelatively low level of gene expression for all genes except forAT3G4725 and AT2G38540 which display higher gene expression in stages2-9 as can be seen from the Percent of Expression Potential (FIG. 7A andFIG. 7B). An overview of all ATH experiments in 127 anatomical parts ofArabidopsis displays moderate gene expression for the listed PF3140genes in all anatomical parts with the exception of the non-detectedexpression for AT3G47250 in roots and extremely low gene expression ofall genes except for AT3G47250 in the abscission zone (FIG. 7C). Thenext experiment included the available four datasets for which geneexpression in young and developed flower is described excluding sampleswith external perturbations in the experiments and curated for wild typeArabidopsis samples using the information in the cited literature and/ordatabases used. The selected anatomical parts now display very lowPercent of Expression Potential in flowers for the listed PF3140 genesexcept for AT3G47210, AT3G47250 and the ATH TDF1 control (FIG. 9A). Adetailed view of gene expression in early and late flowers displays nogene expression for five gene models taken into account that the numberof experimental data was limited yet significant (FIG. 9A). Therecalculated absolute expression levels in both stamen and pistilsamples display the same result (data not shown). HierarchicalClustering (Pearson correlation indices) of both anatomical parts andPercent of Expression Potential has high correlation values for thecluster {AT3G50130, AT3G50140, AT3G50190}, cluster {AT3G50150,AT3G50160, AT3G50120, AT3G50180} and unrelated cluster {AT3G250,AT2G38540} (FIG. 9C). In conclusion, careful mining of curatedArabidopsis gene expression data doe selected DUF247-like genes inGENEVESTIGATOR interfaces for correlation in gene expression indevelopmental stages and 127 anatomical parts displays three highlycorrelated clusters with virtually no gene expression in flowerscompared to other organs for two clusters.

Moreover, the ATH TDF1 gene is expressed at high levels across allstages of development in a systemic way. One can speculate thatDUF247—being a dominant Female suppressor gene, should indeed bevirtually inactive in hermaphroditic Arabidopsis flowers whereasrecessive Male promoting genes such as ATH TDF1 should be active inearly and late flower development. It was decided to investigate whetheror not mutations in the AT3G50150 gene and as such would give clearphenotypical differences compared to the non-mutated phenotype inferringa function of DUF247 in inflorescence development of Asparagusofficinalis.

For this, the Nottingham Arabidopsis Stock Centre (N ASC, University ofNottingham, Loughborough, United Kingdom) was investigated for theavailability of sequence-indexed mutant lines of the listed DUF247 genemodels. For all gene models, germplasm of mutated lines could be madeavailable by the NASC. It was investigated if the allele type i.e. aclassification of alleles based upon the phenotype and genotype ofalleles stated was known. The result being that none of the listed lineshas been given reliable allele type and phenotype description in theTAIR and related databases such as NASC and AtGDB(http://www.plantgdb.org/AtGDB/). This was verified at the SALKInstitute for the insertion lines indicated by the prefix SALK_ andindeed no allele type was available (J. Ecker, Salk Institute forBiological Studies, La Jolla Calif., USA). The six lines indicated‘investigated’ were visually inspected on growth in general, floweringtime, inflorescence architecture and spikelet formation. For this,typical experiments of −50 seeds of the lines, including the Col-0genotype (Species Variant: 90) were briefly sterilized and plated onsolid MS1 medium, placed for 24 hrs. in the dark at 4° C. and forgermination under sterile conditions 10-15 days in growth chambers undercontinuous light, 23° C. The seedlings were transferred to soil andafter 10 days their growth was monitored. For the genotypes indicated noclear phenotypes differentiating from the Col-0 background could beobserved. For a subset of SALK_109348.55.50.X (AT3G50150), SALK_122060(AT3G50160) and SALK_009839 (AT3G47200) flowers architecture was studiedmicroscopically. The result being that in preparations of flower buds instage 8-13, no relevant differences of flower anatomic parts compared toCol-0 background was observed (in collaboration with WUR, Dept. ofBiochemistry, Wageningen, The Netherlands). It was concluded that uponvisual inspection of six lines of DUF247-like Arabidopsis genes no clearphenotypical differences compared to Col-0 background could be observedthereby not inferring a biological function of DUF247 in inflorescencedevelopment of Asparagus officinalis. Further investigation of allhomozygous mutant lines indicated will be performed using DigitalPhenotyping in the KeyBox (KeyGene, Wageningen, The Netherlands).

TABLE X Arabidopsis thaliana genes with significant identities toAsparagus officinalis DUF247-like gene. Indicated is the gene ID (AGICode), predicted subcellular location (SUBA) and NASC ID and informationof mutant lines for the genes. See text fordetails. AGI Code SUB3A NASCID TAIR Name Gene Model(s) Genotype Allele mutagen AT3G50150mitochondrion N673170 SALK_109348.55.50.X AT3G50150.1 Homozygous T-DNAinsertion investigated AT3G50160 mitochondrion N622060 SALK_122060AT3G50160.1 segregating T-DNA insertion AT3G50170 peroxisome N509186SALK_009186 AT3G50170.1 Homozygous T-DNA insertion investigatedAT3G50120 Plasma N675592 SALK_065145C AT3G50120.1 Homozygous T-DNAinsertion investigated membrane AT3G50140 plastid N660145 SALK_122700CAT3G50140.1 Homozygous T-DNA insertion AT3G50130 plastid N470472GABI-KAT 735A08 AT3G130.1 segregating T-DNA insertion AT3G50190 plastidN627332 SALK_127332 AT3G50190.1 segregating T-DNA insertion AT3G50180plastid N677496 SALK_151411C AT2G50180.1 Homozygous T-DNA insertionAT3G47200 plastid N509839 SALK_009839 AT3G47200.1 Homozygous T-DNAinsertion investigated AT3G47200 N509839 SALK_009839 AT3G47200.2Homozygous T-DNA insertion investigated AT3G47250 cytosol N673179SALK_110471C AT3G47250.3 Homozygous T-DNA insertion AT3G47250 N673179SALK_110471C AT3G47250.2 Homozygous T-DNA insertion AT3G47250 N673179SALK_110471C AT3G47250.1 Homozygous T-DNA insertion AT3G47210 plastidN657798 SALK_121894.11.20X AT3G47210.1 Homozygous T-DNA insertioninvestigated

As long as no clear phenotype has been observed in Arabidopsi this meansthat these DUF domain comprising Arabidopsis genes can be considered tobe homologues to the GDS DUF247 gene of SEQ ID NO: 1.

It was decided that the ML4 DUF247-like gene was further investigated inseveral Asparagus genotypes. Dideoxy sequencing (Sanger sequencing) ofwas conducted in the region that includes the predicted DUF247-like geneusing primer pairs designed using Primer 3 (Untergasser, 2007) Theseprimers, referred to as CN59/CN60 CN67/CN68, CN69/CN70, CN71/CN72,CN59/CN70, CN67/CN82, CN69/CN81 are listed in Table 3. We have obtainedsequences of four unrelated male plants DH00/086, 9M, 88M, K323, 12_25and hermaphrodite Herma5375. The prediction in this example starts atthe start codon predicted by the EVM model (see table 6). At nucleotideposition 527 in CDS1, all male plants show a thymine base whereas thehermaphrodite shows a single base pair deletion at this position. Thisdeletion will cause a frame-shift in the reading frame, a change ofamino-acids and after splicing it likely causes a premature stop codon.The amino acids for the hermaphrodite as shown in white text against ablack background and for CDS2 the anticipated premature stop codon isindicated. Besides the structural difference in the exon, unique tohermaphrodite 5375, two SNPs are found in the first intron in 9M and oneSNP in CDS2 that is a synonymous substitution (a silent mutation thatdoes not result into an amino-acids change). 12_25M, K323 and Herma 5375show a single base pair INDEL in the predicted intron compared to theother sequenced males. In CDS3, no differences were found for thesamples for which sequence data was available; DH00/086, K323, and Herma5375. In addition, the results from the aforementioned second RNA-Seqexperiment were included in the investigations of ML4 DUF247. Thegenotypes and their related samples selected for RNA-Seq analysis werethe following: DH Male 1770=sample 1; DH Female 1800=sample 2; Herma5375=sample 3; 5 plants AAff Herma of Pedigree 1E=Bulk 1 and 4 plantsAaFf Males of pedigree 3E=Bulk 2. From each plant, three flower buttonstages were sampled: A) pre-meiosis (1.0-1.2 mm long for Herma and Male,0.8-1.0 mm for Female); B) uni-nucleated microspores (1.6-1.8 mm), orjust developed ovary (1.2-1.4 mm); C) fully developed carpels (justbefore sepal opening). The resulting RNA-Seq data was aligned to theReference AGS V1.10 using TopHat 2.0.13 (Kim et al., 2014). The ML4DUF247 EVM1 annotation was visually inspected. Firstly, gene expressionis detectable but on average less than 2 Fragments Per Kilobase Of ExonPer Million Fragments Mapped (FPKM). A small number of aligned readsfrom the Male Bulk 2 and Male 1770 stage C show the same sequence inCDS1 as was obtained from RNA-Seq data from four unrelated Male plantsDH00/086, 9M, 88M, K323, 12_25, including the Thymidine base at position527 in CDS1. Two aligned reads from the Herma Bulk 1 showed the samesingle thymine deletion at position 527 in CSS1 as was obtained for theRNA-Seq data from Hernia 5373. In conclusion, in two separate RNA-Seqexperiments executed with unrelated Male and Hermaphrodite Asparagussamples from flower organs, in all cases, a single base indel atposition 527 of ML4 DUF247 CDS1 was detected causing a premature stopcodon in the mRNA of ML4 DUF247.

To confirm the EVM annotation of the DUF247-like gene, expression wasstudied by isolating total RNA from flower buds of DH00/86 (the plant ofthe asparagus references sequence) and two other non-related plants.Total RNA was isolated using the RNeasy® Plant Mini Kit (Qiagen)according to the RNeasy Mini handbook (Qiagen) using 15 mg fresh youngflower buds from asparagus and elder flowers which were completelyopened, which were ground in liquid nitrogen. To avoid RNA degradation,RNase-free disposables and 0.1% DEPC treated pestles and glassware wasused. Prior to cDNA synthesis, RNA was treated with DNase I (SigmaAldrich) according to the manufacturer's protocol. Subsequently, cDNAwas synthesized by using Maxima Reverse Transcriptase (ThermoScientific) using 2 μl total RNA, 1 μl (200 U) Maxima ReverseTranscriptase, 100 pmol oligo(dT) primer, 0.5 mM dNTP mix (10 mM each),5× RT buffer and RNase-free water in a final volume of 40 μl. Themixture was incubated for 30 minutes by 50° C., followed by inactivationat 85° C. for 5 minutes.

Following DNAse I-treatment according to the manufacturer's protocols(Thermo Scientific, Pittsburgh, Pa., Sigma-Aldrich, St. Louis, Mo.) theRNA quality was assessed on agarose electrophoresis and Agilent RNABioanalyzer protocols (Agilent, Santa Clara, Calif.). Subsequently,first strand cDNA was synthesized by using Maxima Reverse Transcriptase(Thermo Scientific Pittsburgh, Pa.) using 2 μl total RNA, 1 μl (200 U)Maxima Reverse Transcriptase and 100 pmol oligo(dT) primer. Specific PCRproducts were amplified using primers targeted at predicted exonpositions (Table 3) using the prepared first-strand cDNA as template ina PCR using Phire Hot Start II DNA Polymerase (Thermo Scientific,Pittsburgh, Pa.). As control samples, genomic DNA was included asseparate PCR templates. Primers pairs CR55/CR57, CP35/CR57, CP45/CR57,CP61/CP40, CP61/CR56, CP33/CP38, CP33/CP40 all yielded single PCRproducts which had sizes that corresponded well with gene predictions,as inferred from their migration on a 1,5% agarose gel compared to theGeneRuler 100 bp Plus DNA Ladder (Thermo Scientific, Pittsburgh, Pa.).Compared to the cDNA template the genomic control template alwaysyielded longer fragments of expected sizes. Primer pairs CP61/CP62,CP33/CP62 failed to amplify any products on cDNA template, whereasgenomic DNA template yielded fragments of expected sizes. For PCRproducts of CR55/CR57 and CP35/CR57 on first strand cDNA of the batchtotal RNA from flower buds in several developmental stages of DH00/086,both forward and reverse sequence reads were obtained by directsequencing at BaseClear (Leiden, The Netherlands). The alignment ofthese 4 sequences showed that the 5′-splice site in AUGUSTUS and EVM1annotations for the boundary of CDS2/Intron2 is not correct. In fact,the Cytosine at position 2795 in the generic sequence of ML4 has neverbeen observed in Arabidopsis splice data (Szcześniak et al., 2013). Thenew splice site has the 100% preserved Guanine-Thymidine dinucleotide atpositions 2834-2835 in the generic sequence of ML4. As a result a newstop codon is introduced (TGA) at positions 3616.3618 in the genericsequence and hence CDS3 is only 27 hp. The final spliced sequences forDUF247 EVM1 and for DUF247_DH as well as their respective translationsare shown in FIG. 10. To address the 3′-Untranslated sequence of ML4DUF247-like transcripts, a Rapid Amplification of cDNA Ends (3′-RACE)was designed. For this, the batch total RNA from flower buds in severaldevelopmental stages of DH00/086 was used. First strand cDNA wassynthesized by using Maxima Reverse Transcriptase (Thermo ScientificPittsburgh, Pa.) using 2 μl total RNA, 1 μl (200 U) Maxima ReverseTranscriptase and 100 pmol adaptor oligo(dT) primer(5′-GACCACGCGTATCGATGTCGACTFITTTTTTTTTTTTTTTTTVN) (SEQ ID NO: 16). Thefirst strand cDNA was used for a linear PCR using forward primers CP39and CP35. The products of these linear PCRs were diluted and used astemplate of a nested PCR, using CP41 (downstream CP39 and CP39(downstream CP35) and a universe primer complementary to the tail of theadaptor oligo(dT) primer. After electrophoresis, The two PCR productswere excised from an agarose and send for sequencing to Baseclear(Leiden, The Netherlands).

TABLE 3 primers used in Example 1. SEQ ID NOS CN59DUF 247 M locus scaffold 4 AAATTCTGCAAGAACAAGGTAAGG (17) CN60DUF 247 M locus scaffold 4 TACTGCAAAATTATGGTGAGCATT (18) CN67duf exon 1 fw CTTCGAGCTCCCTTCTCAAA (19) CN68 duf exon 1 RvTCAATCATGAAAGCCCCATC (20) CN69 duf exon 2 fw TAAAGCTATCGTAATTTTATGCTGT(21) CN70 duf exon 2 Rv TCAAATGCTTCGTCAACGTC (22) CN71 duf exon 3 fwATGGCGAAGGTCAAGAGGTA (23) CN72 duf exon 3 Rv TGCCATAGATTGTTTGAGTGATG(24) CN73 duf upstream Fw TAGATGAATCCCGGCCTTG (25) CN74 duf upstream RvTTGCAACAAGCCCATAAAAA (26) CN78 DUF247 forward CATAAGCCATCAACGTGCAG (27)CN81 duf exon 3 new reverse AGTTGAGTTCAGGGTGTGGA (28) CN82duf exon 1 new reverse AGGTTAATCTTGCATTACGAGGT (29) CN83gamma_1R points to the gene GCTCCGGCATTATCAAAGAG (30) CN84gamma_2R points to the gene CCGGCATTATCAAAGAGAGC (31) CP31DUF 247 scanning exon 1 pair 1 AGCCTGGGTTTCTCGATTGA (32) CP32DUF 247 scanning exon 1 pair 1 CCTCAGGGCTCGTATGATGT (33) CP33DUF 247 scanning exon 1 pair 2 TCCTCATCCGATGTCAAGTG (34) CP34DUF 247 scanning exon 1 pair 2 CGACCAAGTATGGCTTCTTGA (35) CP35DUF 247 scanning exon 1 pair 3 ATCATGCCAAGGACCCAATA (36) CP38DUF 247 scanning exon 2 pair 1 ATGACAGCGTTTCACTGCAC (37) CP39DUF 247 scanning exon 2 pair 2 CTGTCATTGACAGATATATGCTTCA (38) CP40DUF 247 scanning exon 2 pair 2 TGCAACTATACCTTTTGTCAGTCC (39) CP41DUF 247 scanning exon 2 & CN72 GTCGGGGGTAAGCAGTGATA (40) CP45DUF 247 primer AGAAAACAGTGGAATTGCG (41) CP61 DUF247 cDNA primer Fw 1ATGGCGGAGGCCTGGA (42) CP62 DUF247 cDNA primer RvTTAACTACACTTATTATAAGAAAGGATG (43) CR37 dCAPS primer Hpy188III TGGGCGGGCAGGTTGGATAATCAAATTTCAA (44) deletion 5375 CR38dCAPS primer Hpy188III T ACAGCTGGGACATTTCAAGG (45) deletion 5375 CR39DUF T deletion HRM marker CTCAGGTTGGATAATCAAATTCCA (46) CR40DUF T deletion HRM marker AGACAATATCTCCAGGACCTT (47) CR55EVM prediction check DUF247 Fw ATGTCTGAAGCCTGGGTTTC (48) CR56FGENESH check DUF247 exon 2 Rv TTACCCATGGATTCGCAAAG (49) CR57EVM exon 3 check 247 Rv TGTTCTCAAGCCACAAACAA (50) CK63 Asp_80-HRMTCTGGCACTAAGAATCAGTTCCT (51) CK64 Asp_80-HRM GCGAGTTTCCAACGAAATTA (52)CP80 DUF247 exon SNP-HRM TTATACAGCATGGAGGTTATCATCACA (53) CP81DUF247 exon SNP-HRM CGATAGTGGTTGGCGAC (54) CM45comp49320_c1F2 :Zf- AN1 HRM GCAGTTGTTGATGCAGAGGA (55) CM46comp49320_c1R2 :Zf- AN1 HRM GAAACAATGGAGCACCACAA (56) CS77bisulfite primer pair 2F TGGATGAAGAATGATGATGAGTTT (57) CS78bisulfite primer pair 2R TTTCATTAACATTCCTTACCTTATTCT (58) CN96905 scaffold start HRM GTGAGCTTAGGGCTTATGTT (59) CN97905 scaffold start HRM CATCTTCTCATAATGACCCAAATATTT (60) CQ31scaffold 2312 ATGGATTCGACTCGGAGACT (61) CQ32 scaffold 2312 TGAGTTGAGAGGGTGGAGGA (62) CT13 scaffold 206 Asp448 like for K1036 newAGGAAATTTTGCACTCAAAGGTA (63) CT14 scaffold 206 Asp448 like for K1036 newGCTTCTGTTGCAGTGCA (64) CE40 Asp448 fw BseNI CAPS markerGTTGCAGTGCAGAAGACCAA (65) CE41 Asp448 Rv BseNI CAPS markerGAACAGGGGCATTTGACAGT (66) CE64 contig04556 CTCAAGGGGCTTGTTTGTTC (67)CE65 contig04556 CGTTTATGGGTTGGACCACT (68) CR61DUF 247 scanning exon 3 pair 1 TGTGCTTAATTTCGCTTCTCCACT (69) CT72Scaffold 1204 HRM for Peru deletion mut GCTGGAATTGATTACTTCGCC (70) CT73Scaffold 1204 HRM for Peru deletion mut GATGAGAGTCGCGAGACAC (71) CE64M-locus HRM CTCAAGGGGCTTGTTTGTTC (72) CE66 M-locus HRMGCCACGGCCTAGTTTAAGAA (73) CT33 DEFECTIVE IN MERISTEMTCATCCAATGTGGTGCTTGT (74) DEVELOPMENT AND FUNCTION F3 CT34DEFECTIVE IN MERISTEM CCATATCCATTCACCACCAA (75)DEVELOPMENT AND FUNCTION R2 CT33 DEFECTIVE IN MERISTEMACCCTCCACCCTTCAACAC (76) DEVELOPMENT AND FUNCTION F3 CT34DEFECTIVE IN MERISTEM CCATATCCATTCACCACCAA (77)DEVELOPMENT AND FUNCTION R3 CL44 scaffold 1194-HRMGTCCTGCAGATAAATTAAGTGCGT (78) CL45 scaffold 1194-HRMTCAGGTCTACTAATACTCAAACAGCT (79) CM98 Asp_446_HRM scaffold 1539GGTAGTTTTGTAGGGCCCA (80) CM99 Asp_446_HRM scaffold 1539AAAAGGCACCAAATTTAAGGC (81) CL83 ARM HRM Marker on scaffold 945GATGTCCACCAAACTTTCTAGCT (82) CL84 ARM HRM Marker on scaffold 945TGGCTGAATAAAACTTGTGTCAA (83) CK33 Asp_432-HRM GCCTCGAAAGCTCTTCTTCT (84)CK34 Asp_432-HRM TGCATAAGCAGTAACTCCAAACA (85) CN94Tapetum related gene scaffold 905 ATTAAGCCTAACTATCAAAATAGTCCAA (86) CN95Tapetum related gene scaffold 905 ACCTATCAGCTGAGAAATTCAATG (87)

These results demonstrate that the DUF247 like-gene is expressed inflower buds and that the expressed gene sequence of the hermaphrodite(at least) differs by a single nucleotide deletion from the genesequence of male plants. It was already mentioned that the gene wasfound in close proximity to published sex linked markers. In order todemonstrate linkage of the mutation itself and the hermaphrodite flowertrait we have analyzed several plants of pedigree Cross 3E. We have usedprimer pair CR39/CR40 (Table 3) in a High Resolution Melting Curveanalysis, which essentially follows the method described in Wittwer etal (2003). Results are shown in Table 1e. The results show fullco-segregation of the marker and the hermaphrodite trait. All twelvehermaphrodites have the marker allele diagnostic for the thyminedeletion whereas all twelve male plants had the wild type gene allele.This confirmed that the single hermaphrodite plant, that was previouslydescribed to have an unexpected 166/166 AO022 microsatellite markergenotype indeed resulted from a recombination event between the 166allele and the gynoecium development repressor gene as also this plantshows the CR39/CR40 marker genotype diagnostic for the single base pairdeletion and must have the ‘Aaff’ genotype. This results was confirmedusing a dCAPS marker using primers pairs CR37/CR38 and the restrictionenzyme Hpy188III. These markers thus are suitable for detection of thisspecific deletion mutant and can as such be used in diagnostic andbreeding methods described in this application. Based on the evidencedprovided above it was concluded that the DUF247-like gene is theGynoecium Development Supressor (GDS) gene,

In general, it can be said that many of the markers that are mentionedin the present application may be suitable to indicate the presence of amutation in the GDS gene or near the GDS gene and/or are suitable toindicate the presence of the allele of the GDS gene. Preferably suchmarkers target the GDS gene, its mutants or alleles or 5′UTR or 3′UTR orits cis regulatory elements. However, other markers can also be used tosuitably indicate the presence of a mutation in the GDS gene or near theGDS gene and/or are suitable to indicate the presence of the allele ofthe GDS gene, when these markers, genetically linked to the GDS gene candisclose polymorphism(s) in a plant that has been shown to have amutation in or near the GDS gene that will cause reduced functionalexpression of the GDS gene. All such markers thus could advantageouslybe used in marker assisted breeding. Primer pairs that can be used fordetection of a mutation may be selected from the group of CN67/CN68,CN69/CN70, CN71/CN72, CN59/CN70, CN67/CN82, CN69/CN81, CP31/CP32,CP33/CP34, CP35/CP36, CP37/CP38, CP39/C40, CP41/CN72, CR61/CR57,CP35/CR57, but other combinations of these primers and/or with otherprimers mentioned in Table 3 will be possible.

Further, markers that are located near to the GDS gene locus and whichmay be used in marker assisted breeding are, next to the ones thatalready have been mentioned above, such as AO022 and Asp 1-T7, listed inTable 5.

TABLE 5 Markers that can be advantageously used inmarker assisted breeding for the GDS gene. SEQ ID NOS: CK63 Asp_80-HRMTCTGGCACTAAGAATCAGTTCCT (88) CK64 Asp_80-HRM GCGAGTTTCCAACGAAATTA (89)CK33 Asp_432-HRM GCCTCGAAAGCTCTTCTTCT (90) CK34 Asp_432-HRMTGCATAAGCAGTAACTCCAAACA (91) CE40 Asp448-BseNI GTTGCAGTGCAGAAGACCAA (92)CE41 Asp448-BseNI GAACAGGGGCATTTGACAGT (93)

It has thus been shown that an exceptional hermaphrodite plant has beenobtained following tissue culture, which is more capable of producingberries than any of its known male ancestors, that has a singlenucleotide deletion in a gene, now designated a Gynoecium DevelopmentSuppressor gene, located on a hemizygous region that was targeted bypublished genetic markers. Further, it has been shown that a GDS genehaving this single nucleotide deletion co-segregates with the plantsthereby maintaining the hermaphrodite phenotype. The tissue culturemethods that has been applied essentially follows the method publishedby Qiao & Falavigna (1990) Briefly; an anther is grafted in an embryoinduction medium that contains 2,4D, an embryo-like structure (a ball of1 mm diameter) is obtained that is transferred to a next medium designedto generate callus from which shoot sprout, these shoots are choppedinto pieces to allow new shoot formation from axillary meristems,finally shoots are placed on a rooting induction medium to obtain rootedmini-crows that can finally transferred to the greenhouse. Since the1980's of the previous century it has been recognized that tissueculture of plants poses the risk of somaclonal variation (Evans et al1984). Somaclonal variation may include point mutations (Jiang et al2011) Somaclonal variation has been recognized as possibility recover ofnovel genotypes. (Evans & Bravo, 1986) Somaclonal variation has beendescribed to result in phenotypic variants of asparagus including plantsshowing differences in flower morphology. Pontaroli & Camadro (2005)compared plant height, cladode length and shape, foliage color, of therespective donor clones and their regenerants. These authors obtainedregenerants of which one was which was greenish blue (glaucous) ratherthan green as the donor and all the other regenerants. More importantlyto our example, Pontaroli & Camadro (2005) obtained regenerants withaberrant flowers with a higher than normal number of stamens of whichsome were adhered to the tepals, some tepals also being fused with theterminal cladodes.

The particular mutation observed in 5375 likely resulted from somaclonalvariation that could potentially occur in the anther culture followingthe method of Qiao & Falavigna (1990) because the plants from whichtissue was taken to start the culture have not shown a hermaphroditephenotype.

As will be pointed out in EXAMPLE 6 and EXAMPLE 2 the GDS gene islocated in a chromosome region hemizygous in males that is absent infemales. The result of this is that a single loss of function allele ofthe GDS gene, if it occurs in vivo in a heterozygous male, will not bemasked by another wild type allele of the GDS gene and has a lowprobability to be left unnoticed.

TABLE 6 Coding sequences (CDS) of predicted exons DUF247 FG(FGenesh prediction) and DUF247 EVM (Evidence Modeler prediction)and detected cDNA sequences (DUF247 DH). Below are their respectiveconceptual translations of the CDS structures ML4ATGTCTGAAGCCTGGGTTTCTCGATTGACATCGGATATAGGGTGGCTCAATAGC DUF247ACAAATGCCCTGATGGCGGAGGCCTGGAGTCGTCATTCAATCTACGACGTACCA EVM CDS1GACACATTCAAAAGGATTAGCCCACAGATCCATAAGCCATCAACGTGCAGCATT 768 . . . 1334GGACCACGGTACAATGGAGATCTGAATCTCCTTCGTATGGAACGTCATAAACAC SEQ ID NO: 94AGGGCGCTACTGAACTTCCTCATCCGATGTCAAGTGTCGATCCATGACATCATACGAGCCCTGAGGAAGAACCTGCACGATTTCAGAGCCTGCTATCAAGATCTTGACACCTTTTGGATGAAGAATGATGATGAGTTCCTAAAAATCATGATTTACGATGGGGCTTTCATGATTGAAATCATGATAGCGACCGTTGAACCATATGAGCGCACACCTTCTAGCTATCATGCCAAGGACCCAATATTCAAGAAGCCATACTTGGTCGAAGATCTTCGTGTAGATATGCTCAGGTTGGATAATCAAATTCCAATGAAGGTCCTGGAGATATTGTCTAAATTCTGCAAGAACAAG ML4ATCCAAAGCATTCATCAGCTGATCAGACATTTCTTCTTCCGCAAATATGAAGAG DUF247GGAAGATATGATATTAGCCAAACCTCTACGATATTTCACCTACCCGAGATAACA EVM CDS2GGGCATCACCTACTGGATGTGTACAAAAAAACTCTTATACAGCATGGAGGTTAT 1798 . . . 2390CATCACACCAGCAGTCGCCAACCACTATCGGCAGTTGAACTACAGGAGGCGGGC SEQ ID NO: 96GTAATTTTCCAGTGCAGTGAAACGCTGTCATTGACAGATATATGCTTCACCAAAGGTGTCCTTTGCCTACCTGCAGTCGACGTTGACGAAGCATTTGAAGTTGTTATGCGGAATCTCATTGCCTATGAGCAAGCACATGGCGAAGGTCAAGAGGTAACATCCTATGTGTTTTTTATGGATGGCATTGTAAACAATGACAAAGATATTGCCTTGCTTCGAGAGAAGGGTATTATCAGGTCGGGGGTAAGCAGTGATAAGAGGATAGCCGATCTTTTTAATGGACTGACAAAAGGTATAGTTGCAAAAGTTGTCGACAATGTTGATGTTGATGTAACCAAGGACATCAATGAGTATTGCAATAGAAGATGGAACAGGTG ML4GCCTTTCCCGGGATTCATAAATGTTGATCTCAACGGTAGGGTTTCGTGCTGGGG DUF247TTTGAGTATCTGTGGAGCATTTAGTGTGAGAAAACTGTGCTTAATTTCGCTTCT EVM CDS3CCACTATGAGAGTGGAGGAGCACAACTAATGGTATCCAGTGTAAATTTAACTCT 3189 . . . 3387TTGTTTGTGGCTTGAGAACAACATGTTCTTTATATAG SEQ ID NO: 98 ML4ATGTCTGAAGCCTGGGTTTCTCGATTGACATCGGATATAGGGTGGCTCAATAGC DUF247ACAAATGCCCTGATGGCGGAGGCCTGGAGTCGTCATTCAATCTACGACGTACCA DH CDS1GACACATTCAAAAGGATTAGCCCACAGATCCATAAGCCATCAACGTGCAGCATT 768 . . . 1334GGACCACGGTACAATGGAGATCTGAATCTCCTTCGTATGGAACGTCATAAACAC SEQ ID NO: 100AGGGCGCTACTGAACTTCCTCATCCGATGTCAAGTGTCGATCCATGACATCATACGAGCCCTGAGGAAGAACCTGCACGATTTCAGAGCCTGCTATCAAGATCTTGACACCTTTTGGATGAAGAATGATGATGAGTTCCTAAAAATCATGATTTACGATGGGGCTTTCATGATTGAAATCATGATAGCGACCGTTGAACCATATGAGCGCACACCTTCTAGCTATCATGCCAAGGACCCAATATTCAAGAAGCCATACTTGGTCGAAGATCTTCGTGTAGATATGCTCAGGTTGGATAATCAAATTCCAATGAAGGTCCTGGAGATATTGTCTAAATTCTGCAAGAACAAG ML4ATCCAAAGCATTCATCAGCTGATCAGACATTTCTTCTTCCGCAAATATGAAGAG DUF247GGAAGATATGATATTAGCCAAACCTCTACGATATTTCACCTACCCGAGATAACA DH CDS2GGGCATCACCTACTGGATGTGTACAAAAAAACTCTTATACAGCATGGAGGTTAT 1798 . . . 2430CATCACACCAGCAGTCGCCAACCACTATCGGCAGTTGAACTACAGGAGGCGGGC SEQ ID NO: 102GTAATTTTCCAGTGCAGTGAAACGCTGTCATTGACAGATATATGCTTCACCAAAGGTGTCCTTTGCCTACCTGCAGTCGACGTTGACGAAGCATTTGAAGTTGTTATGCGGAATCTCATTGCCTATGAGCAAGCACATGGCGAAGGTCAAGAGGTAACATCCTATGTGTTTTTTATGGATGGCATTGTAAACAATGACAAAGATATTGCCTTGCTTCGAGAGAAGGGTATTATCAGGTCGGGGGTAAGCAGTGATAAGAGGATAGCCGATCTTTTTAATGGACTGACAAAAGGTATAGTTGCAAAAGTTGTCGACAATGTTGATGTTGATGTAACCAAGGACATCAATGAGTATTGCAATAGAAGATGGAACAGGTGGCAAGCCAACTTTAAGCAGAGATACTTTGCGAATCCATGG ML4 GCCTTTCCCGGGATTCATAAATGTTGADUF247 DH CDS3 3189 . . . 3215 SEQ ID NO: 104 ML4ATGGCGGAGGCCTGGAGTCGTCATTCAATCTACGACGTACCAGACACATTCAAA DUF247AGGATTAGCCCACAGATCCATAAGCCATCAACGTGCAGCATTGGACCACGGTAC FG CDSfAATGGAGATCTGAATCTCCTTCGTATGGAACGTCATAAACACAGGGCGCTACTG 834 . . . 1334AACTTCCTCATCCGATGTCAAGTGTCGATCCATGACATCATACGAGCCCTGAGG SEQ ID NO: 106AAGAACCTGCACGATTTCAGAGCCTGCTATCAAGATCTTGACACCTTTTGGATGAAGAATGATGATGAGTTCCTAAAAATCATGATTTACGATGGGGCTTTCATGATTGAAATCATGATAGCGACCGTTGAACCATATGAGCGCACACCTTCTAGCTATCATGCCAAGGACCCAATATTCAAGAAGCCATACTTGGTCGAAGATCTTCGTGTAGATATGCTCAGGTTGGATAATCAAATTCCAATGAAGGTCCTGGAGATATTGTCTAAA TTCTGCAAGAACAAGML4 ATCCAAAGCATTCATCAGCTGATCAGACATTTCTTCTTCCGCAAATATGAAGAG DUF247GGAAGATATGATATTAGCCAAACCTCTACGATATTTCACCTACCCGAGATAACA FG CDS1GGGCATCACCTACTGGATGTGTACAAAAAAACTCTTATACAGCATGGAGGTTAT SEQ ID NO: 108CATCACACCAGCAGTCGCCAACCACTATCGGCAGTTGAACTACAGGAGGCGGGCGTAATTTTCCAGTGCAGTGAAACGCTGTCATTGACAGATATATGCTTCACCAAAGGTGTCCTTTGCCTACCTGCAGTCGACGTTGACGAAGCATTTGAAGTTGTTATGCGGAATCTCATTGCCTATGAGCAAGCACATGGCGAAGGTCAAGAGGTAACATCCTATGTGTTTTTTATGGATGGCATTGTAAACAATGACAAAGATATTGCCTTGCTTCGAGAGAAGGGTATTATCAGGTCGGGGGTAAGCAGTGATAAGAGGATAGCCGATCTTTTTAATGGACTGACAAAAGGTATAGTTGCAAAAGTTGTCGACAATGTTGATGTTGATGTAACCAAGGACATCAATGAGTATTGCAATAGAAGATGGAACAGGTGGCAAGCCAACTTTAAGCAGAGATACTTTGCGAATCCATGGGTAACTTGCTCACTCATTGTAGGAGCTCTAGTATTAGGTCTCACCATCACTCAAACAATCTATGGCATC CTTT

Example 2

Genetic Analysis of Hermaphrodite Mutant K323-G33

All male hybrid K323 is a cross between female doubled haploid LIM425obtained from an anther culture of the cultivar Gladio and a maledoubled haploid L1M428 obtained from an anther culture of the cultivarGijnlim.

LIM428 was selected as parental plant because it, among other criteria,was not capable of producing berries. Although male hybrid K323 has arudimentary style in the gynoecium, and despite of the fact that itsgrandfather Gijnlim sometimes harbors andromonoecious plants, it nevershowed a single berry in more than 15,159 plants in various hybridtrails that were evaluated in the period 1998-2007. It was decided tocreate a mutant version of K323 that acquired the hermaphrodite trait asthe result of a changed GDS mediated by irradiation mutagenesis. Thedecision to provide another example in which a mutation in a GDS resultsinto a plant with the sex linked hermaphrodite trait was made becausethis hybrid poses excellent starting material for mutagenesis. The firstreason is that this hybrid has no tendency at all to produce berrieseven under circumstances that could have favored andromonoecy, such asshort day and cold temperature (Franken, 1970) and plant age for whichthe tendency of andromonoecy peaks at three years (Franken 1970). Suchcircumstance should have occurred during the long period of evaluationof K323 in any case. A second reason is that all plants of this hybridare genetically identical because it results from a cross between twodoubled haploid parents. Therefore any phenotypic change in a plantbelonging to hybrid K323 must be the result of mutation.

To create mutations, 34,000 seeds, obtained by bee pollination in anisolated greenhouse, were exposed to Cobalt-60 gamma irradiation at adose of 450 Gray at the Synergy Health facilities (Synergy Health EdeB.V. Morsestraat 3. Ede, The Netherlands) using their ‘test apparatus’.In this apparatus, the Cobalt-60 source is composed of pencil type rodsthat are arranged concentrically to a cylindrical container in which thesample can be placed. Seeds were provided in petri dishes that werepiled in this container and exposed to the indicated dose of 450 Gray.The dose delivered was measured by a typical dosimetry system thatinvolves the use of Perspex to measure a colorimetric change caused bythe dose.

Irradiated seeds of K323 were sown outdoors in Horst, The Netherlands,in May 2012 and the first flowers were observed and inspected the nextyear from April 2013 until July 2013. It was estimated that roughly halfof the seeds finally provided mature plants meaning that about 17,000flowering plants have been evaluated. A single plant was found thatshowed a stalk bearing berries that appeared to have developed from eachflower. In addition, this plant had a second stalk that produced perfectflowers. Consistent with previous observations, all of the other K323plants did not produce any berry. The single hermaphrodite K323 plant,designated ‘K323-G033’ or shortly ‘G-033’ was transferred to an insectfree greenhouse and further grown in a pot filled with turf where itproduced new stalks all of which showed perfect flowers followed by fullfruit set. An example of the fruit set is shown in FIG. 12-A. Thetypical flowers of K323-G033 compared to flowers of a wild-type (WT)K323 plant grown under similar greenhouse conditions are shown in FIG.12-B. The flowers of G033 have a longer style and better developedstigma lobes that are longer and more curved compared to the WT K323flowers. Later in the season, at short days, which has been shown tofavor andromonoecy in other hybrids (Franken 1970) the most perfectlylooking flowers of WT K323 plants were collected and again compared withG033 to find out if their best developed style and stigma could reachthe level observed in the mutant (FIG. 12-C). The average style lengthof the mutant G033 was 2 millimeters, whereas the \VT K323 plantsmaximally produced styles of 1 millimeter. Clearly, a mutant had beencreated that showed flowers that were more perfect compared to the WTversion of the hybrid and showed full berry set which plants of the WThybrid and its father never did.

The mutant analyzed verified with proprietary microsatellite markerswhich confirmed its expected authenticity; it showed the uniquemicrosatellite profile that is highly discriminative and characteristicfor this hybrid from which this hermaphrodite phenotype has beenobtained (results not shown). It was decided to sequence both Wild typeK323 and its derived hermaphrodite plant K323-G033 in order to comparetheir sequences and to find out which gene mutation caused thehermaphrodite phenotype. The sequences were aligned to a genomereference sequence, which was composed by the laboratory of Dr James HLeebens-Mack, which in collaboration with the Beijing Genomics Institute(BGI) has worked on a draft genome sequence of doubled haploid supermale DH00/086 (version 1.0). in their work sequence reads were mapped toan assembly of 100-90 bp paired end-and mate-pair Illumina sequencesobtained from BGI for a total 163 gigabases of sequence and anapproximate coverage of 123×. The resulting assembly constructed bybioinformaticians at the Beijing Genomics Institute, using a SOAPassembler, exhibited a contig N50 of 21,179 bp and a scaffold N50 of301,040 bp. In other words, half of the genome is assembled in 1196sequence scaffolds that are at least 301,040 bp in length.

The Beijing Genome Institute (BGI) further generated nearly 40× genomecoverage of 100nt paired-end Illumina short reads for the DH00/94 femaledoubled haploid, a sibling to the DH00/086 male doubled haploidindividual that was used for genome assembly and annotation.

Short reads from both G033 and WT K323 were aligned to the referencegenome using bwa-mem with default settings (Li and Durban, 2009).Concurrent alignments were produced with Bowtie2, requiring end-to-endread alignments with no soft clipping or split-read alignments allowed

Leaf tissue from G033 and a wild-type K323plant were similarly sequencedin the Leebens-Mack lab, generating roughly 7× whole genome shotguncoverage (Illumina paired-end 100nt reads) for each library. Reads fromboth libraries were aligned to the genome using bwa-mem. Read coverageat every non-transposon genomic feature from the initial BGI annotationsproduced with GLEAN (eg., whole gene, mRNA, individual exon, CDS, UTR)was counted for both libraries using bedtools coverageBed. Under thehypothesis that gamma irradiation induced a deletion in the G033 plant,data were sorted to identify gene features with 0 read support in theG033 plant and ≥5 reads in the K323 plant, then further sorted toidentify gene features with the greatest read coverage differencebetween the two individuals.

By using this method, a variant sequence, potentially greater than 2kilo-base pairs, was identified that was unique to the genome sequenceof G033. A CDS exon positioned on a sex-linked BAC assembly(M-locus_scaffold4) at positions 2201:2926 had 18 aligned K323 reads and0 aligned G033 reads. Read coverage was visualized within a Jbrowsegenome browser instance. There was strong support for a border of thisvariant indicated by bwa-mem soft-clipped reads at a single location in5 reads, shown by arrows at the right side of reads in FIG. 4. The exactsize of the variant is unknown given the lack of read support toidentify the other border (see further explanation below). More than 200kb of surrounding genomic sequence was deemed to be hemizygous(Y-specific) by the presence of DH00/086 read coverage and the lack ofDH00/94 read coverage.

It can be inferred from the Jbrowse visualization (FIG. 4) that in thegenomic region represented by the scaffold M-locus_scaffold 4 (andsimilarly in the genome scaffold 905) an event has taken place thatcaused a lack of reads for mutant hermaphrodite G033 in a region thatoverlaps a large part of the predicted intron, the predicted second exonand in addition a large part of the transcribed sequence that mayinclude a possible third exon (as predicted by EVM) of a DUF247containing gene. The two distinct gene predictions are visualized inFIG. 4. The sequences of these gene predictions may be found in FIGS. 4and 13.

At the left border from the alignment where reads were lacking for G033so called ‘clipped reads’ were found (indicated by arrows in FIG. 4).These reads were retrieved from the library sequence data and theirentire gene sequences makes them ‘split-reads’. In those split reads,one area (left, relative to the part were reads or missing in G0033)shows homology to M-locus_scaffold4 (sequence depicted in FIG. 13),whereas the other area at the right is identical among these reads butconsistently different from the M-locus_scaffold4. Based on these splitreads, a consensus sequence could be made which showed and suggestedthat at the position of M-locus_scaffold4 an insertion has taken placethat replaced the original sequence. The split-read consensus of thisinsert in intron close to the exon 1 side of the intron is:

TCTGCAAGAACAAGGTAAGGAATGTTAATGAAATCTAAATCTTCATACCTTGAAATGTCCCAGCTGTAACTCCAGAAGAACTTGCACAAAATTTTCCTTATTCCTTATTCCTTATTCCTTGCAGTTATATACGTTATAGCGGATC (SEQ ID NO: 110), where the underlined part indicates theinsertion specific part.

Using this underlined part as a query to mine the sequences data of theG033 library, mate pair sequences were identified that provided aconsensus sequence that extended further into the inserted part. Thissequence consensus was:

TNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTTATATACGTTATAGCGGATCCCATCCATCGGCTCCAAAGCTTCGGCCAGCTGTCGAGCAAGACGTTGACGCTGTCTTTGTCGTGCTCTCTTTGATAATGCCGGAGCGTCTTCAGAAGTC (SEQ ID NO: 111), where the N's denote unknownbases.

Two reverse primers, designated CN83 and CN84 (see primer Table 3) weredesigned that anneal to the sequence of the alleged insertion and pointtowards the first exon. These primers combined with the exon 1 specificprimer CN78 were tested in a PCR to confirm that all short sequencescollected so far for G033 indeed provide a correct representation of theborder of an insert. The template sequences that were used were thesequences of K323-WT, hermaphrodite G033, and DH00/086 where the lasttemplate represents a sample corresponding to the reference genome. Aunique fragment was obtained for mutant G033 that can be used as agenetic marker (See FIG. 11) that was lacking in the K323-WT plant andin the reference genome sample. The Sanger sequences obtained bysequencing this fragment are shown in the following sequence

(SEQ ID NO: 112) CCTTCGTATGGACGTCATAAACACAGGGCGCTACTGAACTTCCTCATCCGATGTCAAGTGTCGATCCATGACATCATACGAGCCCTGAGGAAGAACCTGCACGATTTCAGAGCCTGCTATCAAGATCTTGACACCTTTTGGATGAAGAATGATGATGAGTTCCTAAAAATCATGATTTACGATGGGGCTTTCATGATTGAAATCATGATAGCGACCGTTGAACCATATGAGCGCACACCTTCTAGCTATCATGCCAAGGACCCAATATTCAAGAAGCCATACTTGGTCGAAGATCTTCGTGTAGATATGCTCAGGTTGGATAATCAAATTCCAATGAAGGTCCTGGAGATATTGTCTAAATTCTGCAAGAACAAGGTAAGGAATGTTAATGAAATCTAAATCTTCATACCTTGAAATGTCCCAGCTGTAACTCCAGAAGAACTTGCACAAAATTTTCCTTATTCCTTATTCCTTATTCCTTGCAGTTATATACGTTATAGCGGATCCCATCCATCGGCTCCAAAGCTTCGGCCAGCTGTCGAGCAAGACGTTGACGCTGTCTTTGTCGTGCTCTCTTTGAT

The alignment of this sequence in the predicted intron is shown in FIG.5. Sanger sequencing indeed proved that the fragment is ‘chimeric’ thuscontaining a sequence known to occur in the predicted intron, followedby a unique ‘downstream’ part that must have resulted from an insertionlike event, probably best referred to as a ‘replacement-insertion’.

Whatever the precise event may be, clearly the G033 mutant plants lacksreads in the predicted intron and the predicted exon 2 and exon 3 of theGDS gene (described in EXAMPLE 1) and thus has a disrupted GDS gene.Study of the read mapping in J-browse teaches that reads of G033 arelacking sequences further downstream the GDS gene. This downstreamregion comprises stretches of repetitive DNA, separated by low or singlecopy regions, which can be inferred from the read mapping of the femalereference DH00/94 (described in EXAMPLE 1) that show some read mappingto certain sub-regions (comprising high copy DNA), disrupted by gaps inthe read coverage because there are no female reads that can map tothese truly unique and male-specific sub-regions of DNA. As expected,this ‘patchy distribution’ of reads is not observed for the reads ofDH00/086 mapped to the same reference. It appears from studying the readmapping in genome browser J-browse that the reads obtained from G033show a patchy read distribution, comparable to the distribution offemale DH00/094, up to position 17,500 in Mlocus-Scaffold 4, whereas theread mapping of the K323-WT shows the typical continuous read mappingcomparable to that of the DH00/086 reference male.

In conclusion, the outlook of the read mapping landscape suggests thatthe end of the missing part that resulted from the‘insertion-replacement event’ in G033 is positioned from the GDS geneintron to a position roughly before 1.8 kb from the scaffold start.Further downstream of this latter position, a comparable depth of readmapping is observed for both G033 and the K323 WT control plant. In theregion spanning the disrupted GDS gene up to the hypothesized end of the‘insertion—replacement event’, three coding sequences can be found asidentified by FGENESH. All three coding sequences have hits using BLASTx(Altshul et al., 1990) against non-redundant proteins such as Integrase,catalytic region; Zinc finger, CCM-type (ABD32582.1), Retrotransposongag protein [Asparagus officinalis] ABD63142.1 and Retrotransposon gagprotein [Asparagus officinalis] ABD63135.1. As these annotations wererelated to transposons, rather than plant genes, it was concluded that,similar to the mutation described for EXAMPLE 1, a hermaphrodite planthad been created by a mutation in the GDS gene.

To further investigate the segregation of the hermaphrodite trait ofG033, several crosses were made. From the offspring the gender wasrecorded and the DNA was isolated for a later study of theco-segregation of the CN78/CN83 marker, indicative for theinsertion-replacement event. Phenotyping was performed by both visualinspection of the flowers (typing these as either perfect/female/male)and by inspection of full berry setting under insect free conditions.The results obtained are presented in Table 4.

TABLE 4 Phenotypic segregation results obtained for three pedigrees(G033 self fertilized, GO33 crossed to a male DH and a female crossed toG033) made by using mutant G033 a-parental plant and their markerresults. ‘Marker present’ means that a PCR fragment generated by primerpairs CN78/CN83 or CN78/CN84 that is diagnostic for thedeletion/insertion event is amplified, thus is present, by usingtemplate DNA for the particular plants studied as has been shown fromFIG. 11 (for further explanation see text). G033 self-fertilzed no Aafffemale hermaphrodite male flowering totals marker present 0 46 0 1 47marker absent 8 0 0 14 22 totals 8 46 0 15 Aaff × AAFF G033 crossed tomale no DH female hermaphrodite male flowering totals marker present 0 010 0 10 marker absent 0 0 14 0 14 totals 0 0 24 0 aaff × Aaff no femalecrossed to G033 female hermaphrodite male flowering totals markerpresent 0 53 0 0 53 marker absent 33 0 0 7 40 totals 33 53 0 7.

The progeny that was obtained from self-fertilization of G033, thatwould have an expected genotype Aaff resulted in 46 hermaphrodites and 8females which significantly differs from the expected 3:1 ratio(p<0.02). An explanatory hypothesis for this deviation is that a numberof plants had not been phenotyped during the growing season, which arelikely females as female plants usually flower later than male plants(Lopez_Anido & Cointry, 2008, p89) and possibly also later thanhermaphrodites. All but one of fifteen of the plants that have notflowered lack the diagnostic marker for the GDS gene mutation, which isconsistent with linkage of the GDS gene to the hermaphrodite trait andthe possible late flowering of the female plants. Further allhermaphrodites had the CN78/CN83 marker, whereas it was lacking in theeight females.

In a second cross, hermaphrodite G033 was emasculated and crossed as amother plant to a male doubled haploid, which could be represented as atype of cross with the genetic constitution Aaff×AAFF. A pedigree of 24male plants was obtained in which the marker diagnostic for the mutationin the GDS gene segregates in a 1:1 ratio (10:14). This is consistentwith results presented in EXAMPLE 1, which again indicate that thedominant allele of the repressor of gynoecium development from the maleparent blocks the previously observed gynoecium development of thehermaphrodite. It can thus be concluded that the hermaphrodite trait isa recessive trait.

In a third cross, a female was pollinated by the hermaphrodite togenerate a progeny of 93 plants comprising 53 hermaphrodites and 33females. This ratio significantly deviates from a 3:1 ratio and if it isassumed, as above, that the seven plants that have not yet flowered arefemale plants this deviation is even more extreme. However, thediagnostic marker for the ‘insertion-deletion event’ in the GDS genefully co-segregates with the hermaphrodite trait which confirms thegenetic model of a dominant gene that allows anther development in apedigree devoid of the dominant gene that suppresses gynoeciumdevelopment.

It should be noted that in all of the above crosses flowers ofhermaphrodites were perfect from all of which berries developed.

In conclusion, it has been demonstrated that a disruption in the GDSgene at a male specific region has been created using irradiationmutagenesis. This mutated gene can be transferred to next generationsand confers Mendelian inheritance of the hermaphrodite trait.

Example 3

Epi-Alleles of the DUF247 Domain Containing Female Suppressor Gene

When breeding line K1036 was seed propagated in an isolated greenhousein which bees were placed for pollination, it was noticed that allplants had produced berries, whereas normally half of the plantsexpected to be male would not produce berries. Several years later, lotK1036 was sown again to unravel the inheritance of the hermaphroditetrait. Sixteen plants were evaluated for flowering and fruit set. Allplants, which all had well developed anthers were capable of fruit setunder insect-free conditions. It was noted that fruit set somewhatvaried among those plants and/or among their branches. Despite of somefailures in fruit set, the number of berries on a plant could reach alevel as high as 95%, which is exceptionally high. As breeding recordsprovided insufficient information on the number of generations breedingline K1036 has been propagated, five K1036 plants were genotyped using aset of thirty proprietary microsatellite markers which are used at aroutine basis to monitor both authenticity and the level of inbreedingof breeding stock. The five hermaphrodites appeared fully homozygous atthirty (hypervariable) loci and were virtually identical (results notshown); four plants were fully identical and one plant differed from theother three plants at only two of the thirty loci for which it differedhomozygously for alternative alleles. The level of homozygosity,observed for K1036 is usually only found for doubled haploids obtainedby anther-culture. In conclusion; the hermaphrodite K1036 representsfully homozygous (syngeneic) inbred material.

Despite of the fact that the fruit set of some plants was as high as95%, not all plants perfectly set fruit and some plants lacked berriesfrom tens of flowers. Because the plants were found to be virtuallysyngeneic, differences in fruit set were initially attributed tonon-uniform growing conditions such as plants shading other plants ontables, plants poorly growing after re-potting, insufficient watering,pollination under warm weather conditions etc. To further analyze thephenomenon of incomplete fruit set, several K1036 hermaphrodites werecrossed with two (line 88 and line 105) female testers. All resulting F1plants obtained by these test crosses produced anthers and all plantswere capable of producing berries under insect-free conditions. Fruitset was scored into classes 1-5 which roughly correspond to 0-20%,20%40%, 40%-60%, 60%-80%, 80%400% fruit set. It was observed that mostF1's were highly hermaphrodite (class 5) as if all F1 hybrids inheritedthe trait that was essentially fully penetrant. However, again somepoorly fruit setting plants were noted among plants within F1 pedigreesand one small F1 progeny stood out for a much lower fruit set (867F1b,that has father plant with ID 215292) as if there might be a heritablefactor different for this particular F1 progeny. The fruit set of the F1testcrosses is shown in Table 31. In another experiment the fruit set ofpedigrees obtained from individual self-fertilized K1036 hermaphroditeswas recorded. These results are shown in Table 32. These results againindicate that families seem to segregate in terms of fruit set and thatthe average fruit set differs between pedigrees. However, when one looksat fruit-set of particular K1036 father plant Ills as the result ofself-fertilization and compares this fruit set with the fruit set of thefemale testcross pedigrees sired by those particular individual K1036father plant ID's, there seems no clear correlation. (see Table 31 andTable 32 for similar 21529× ID's).

TABLE 31 The fruit set, on a scale of the F1 testcrosses derived fromK1036 father plants pollinating a female tester (either line 88 or lin105). Fruit set was scored into classes 1-5 which roughly correspond to0-20%, 20%-40%, 40%-60%, 60%-80%, 80%-100% fruit set (berries) fatherNumber of plant per K1036- fruit set scale 1-5 fruit set F1 ID mother ID1 2 3 4 5 average father 876F1a 88 215290 0 1 1 2 28 4.8 nd 876F1b 88215292 2 1 3 1 2 3.0 1 876F1c 88 215293 0 0 0 0 7 5.0 4 877F1a 105215296 1 0 1 2 35 4.8 nd 876F1e 88 215297 0 0 0 1 53 5.0 4 876F1f 88215299 1 0 0 0 34 4.9 3

TABLE 32 The fruit set, on a scale of the of F1 plants derived fromselfing particular K1036 plants indicated by their plant ID. Fruit setwas scored into classes 1-5 which roughly correspond to 0-20%, 20%-40%,40%-60%, 60%-80%, 80%-100% fruit set (berries Fruit set 1-5 fruit setselfed K1036 ID 1 2 3 4 5 average father 215290 5 2 1 1 0 1.8 nd 2152934 0 6 16 8 3.7 4 215295 3 2 3 4 0 2.7 4 215296 1 2 3 2 11 4.1 nd 2152972 1 0 4 0 2.9 4 215299 3 4 2 2 9 3.5 3

It therefore remained questionable whether the observed variability washeritable or largely controlled by the environment. To further analyzethe genetics, an emasculated hermaphrodite K1036 plant (ID 215297) wascrossed to a super-male (line 88 doubled haploid, designated D1402/504).This cross yielded three F1 hybrids (designated 861F1-124M, 861F1.126M,and 861F1.128M) which were all fully male, which means that those plantsall had fertile anthers and did not produce any berries. This indicatesthat the hermaphrodite trait originating from K1036 is recessive to themale trait of super male DH02/504. The fact that test crosses betweenK1036 and female plants yielded hermaphrodites, whereas a cross withsuper-male plants only yielded males suggested that K1036 might belacking a female suppressor as has been found for the hermaphrodites ofEXAMPLE 1 and EXAMPLE 2. To further investigate this, thehermaphrodite×super-male F1 hybrids were back-crossed to line 88 femaleplants. Pedigree 861BC1d, which was a cross between individual female88-100599 and individual 861F1-124M, was phenotyped for fruit set andflower morphology and genotyped using sex linked markers. Genetically,this is a pseudo-testcross of the type: inbred line-88female×(hermaphrodite K1036 inbred line-88 super male). A total of 91plants could be sex-typed of which 53 were male and 38 werehermaphrodite which is not significantly different from a 1:1 ratio(p>0.11) Marker AO022 has three recombination events in 91 samplestested and marker Asp_80 (for primers see Table 3, EXAMPLE 1) showedonly two recombination events among 85 individuals tested. This showsthat the recessive hermaphrodite trait is linked to the M-locus as hasbeen found for hermaphrodites of EXAMPLE 1 and EXAMPLE 2. As opposed toother pedigrees derived from K1036 which showed some variability infruit set, there was no marked variation among hermaphrodites in thisparticular pedigree 861BC1d; all hermaphrodites showed nearly perfectfruit set and had the maximum fruit set score of ‘5’. All hermaphroditesof pedigree 861BC1d showed a well-developed style that would he scoredas a four or five using the classification of Franken, 1969 p37. In allmales of pedigree 861BC1d, the style was lacking and none of these malesproduced any berries and thus were not even slightly andromonoecious.Examples of the two flower phenotypes that segregated in 861BC1d and arerepresentative for the segregating phenotypes in this population areshown in FIG. 14. In conclusion, the hermaphroditism originating fromK1036 and segregating in pedigree 861F1d was a clear mono-genicrecessive trait, linked to the M-locus.

Because the hermaphrodites of EXAMPLE 1 and EXAMPLE 2 showed an singlenucleotide deletion and a large insertion deletion, respectively, in theM-locus linked GDS gene, a mutation was also expected for the K1036hermaphrodite M-locus GDS allele. Sequencing PCR fragments in bothdirections using K1036 as template DNA and using primer pairs, CN67/68,CN67/CN82, CN59/CN70, CN69/CN81, CN59/CN60 (see Table 3, EXAMPLE 1)however, revealed no unique sequence variation. K1036 shows a GDS genehaplotype that is characterized by a SNP at the third codon position ofa serine amino-acid (AGC to AGT) at the 58th amino-acid of the secondpredicted exon) which is a synonymous substitution (thus a silentmutation which retains the serine amino-acid) that can also be found inbreeding line 9M (for sequence of 9M see FIG. 13) that is male (ratherthan hermaphrodite) indicating that this particular SNP has no impact onsex determination. This particular SNP was later exploited as geneticmarker target (see below).

The M-locus linked GDS gene sequence of hermaphrodite K1036 and male 9Mwere found to be fully identical. Efforts to obtain sequence informationupstream the M-locus-linked-GDS gene for mutations that maydifferentiate the K1036 from 9M haplotypes have failed (results notshown) which likely depends on the nature of this sequence. Three PACBio reads which overlap the unknown region upstream the GDS gene towardsa DUF4283 containing gene located further upstream scaffold 905 wereprovided (results not shown). Information obtained by these PAC BIOreads suggest that the region upstream the GDS gene is highly repetitiveand harbors large AT repeats or large AT-rich repeats interspersed byshort GC-rich repeats which makes it impossible to design primers thatwould allow Sanger sequencing of the sequence upstream the GDS gene(that may include the gene promotor or other cis-regulatory elements)for both K1036 and 9M (results not shown),

Long distance amplification using primers, flanking the ‘sequence gap’provided fingerprint-like patterns or fragments that were not authenticas these fragments were also amplified in PCRs lacking one of the twoprimers initially used as a pair; results not shown. In conclusion, itwas impossible to obtain sequence information upstream the M-locuslinked GDS gene.

To find out whether new efforts to detect sequence variation near theGDS were worthwhile, thus that a mutation in line K1036 indeed should besought in the M-locus linked GDS gene (region), efforts were taken tofurther fine-map the K1036 hermaphrodite trait. To this end, more‘861-BC1 crosses’ of the type: inbred line-88 female×(hermaphroditeK1036×inbred line-88 super male) were made. Optimal greenhouse userequired that the population was downsized by selecting and keeping onlyyoung plants that had a recombination event between microsatellitemarkers AO022 and Asp-80 (for marker details see EXAMPLE 1) which flankthe M-locus linked GDS gene at both sides at a genetic distance of lessthan 5 centi-Morgan. Those ‘marker recombinant plants’ were subsequentlyphenotyped for flowering and fruit set. In addition to markers AO022 andAsp-80, plants were genotyped for their GDS allele by Melting Curvemarker CP80/CP81 (for primers see Table 3). This marker targets the SNPin the second predicted exon of the GDS gene of hermaphroditegrand-parent K1036 which differs from the allele of the othergrand-parent DH02/504, the line 88 super male.

For the populations 861BC1a, 861BC1b, 861BC1c, 861BC1e, and 861BC1f; 22,327, 135, 86, and 33 individuals were grown, respectively, from which 18recombinants between marker AO022 and the GDS gene locus and 8recombinants between the GDS gene locus and Asp_80 were obtained forfurther phenotyping. Those recombinants, together with some ‘controlplants’ (for which AO022, GDS, and Asp_80 did not recombine), provided apanel of 44 plants that was further phenotyped in the next season.Twenty-five of the plants in this panel, which had the GDS alleleoriginating from the DH02/504 male grand-parent, were not capable ofproducing berries. All those twenty-five plants had style lengths thatwould be classified as 1 (no style at all) in the scale of Franken,1969; p37. In conclusion, if a BC1 plant received the DH02/504 malegrand-parent M-locus GDS allele, it was a male plant. Nineteen plants inthe panel had the K1036 hermaphrodite grand-parental M-locus GDS—allele.Among these nineteen plants variability has been observed: Twelve plantsproduced over five-hundred berries and these plants had styles qualifiedas either a ‘5’ (n=9 plants) or a ‘4’ (n=3 plants). Two plants producedroughly 200 berries and had styles classified as ‘5’ or ‘4’. Another twoplants produced roughly 100 berries and had styles classified as ‘3’ and‘2’. Three remaining plants, which had a style classified as only ‘1’,produced only five berries (n=1 plant) or no berries at all (n=2plants). One plant that was incapable of fruit set, which carried theK1036 hermaphrodite grand-parental M-locus GDS allele, was a ‘control’hermaphrodite rather than ‘a marker recombinant’ which indicates thatthe phenomenon of variable fruit set was not per se related torecombination events in the M-locus region but generally occurred inthis population.

In conclusion, in the additional 861BC1 pedigrees no fruit set wasobserved among plants that carry the DH02/504 male grand-parent M-locusGDS allele which were all male (just like in pedigree 861BC1d), whereasamong plants that carry the K1036 hermaphrodite grand-parental M-locusGDS allele, all but two plants set fruit. However, variability wasobserved for the level of fruit set among those plant which set fruit.This fruit set appeared to be related to how well the style wasdeveloped. This situation, observed for the additional 861BC1 (861BC1a-fother than 861BC1d) pedigrees is different compared to the resultsobtained for pedigree 861BC1d, because in the latter population aninvariably high level of hermaphroditism was observed among all plants(thus without exceptions) that carried the K1036 hermaphroditegrand-parental M-locus GDS allele.

In EXAMPLE 1 and EXAMPLES 2 it has been shown that a recessive allelecaused the loss of a normally dominant female suppressor GDS Resultsobtained for pedigree 861BC1d in the present EXAMPLE were consistentwith that model, although the cause of loss of function has not becomeclear. For the other 861BC1 pedigrees, phenotypes have been found whichsuggest incomplete penetrance of the K1036 hermaphrodite grand-parentalM-locus GDS allele which likely must be interpreted as an ‘incompletelylost’ or ‘incompletely suppressed’ female suppressor GDS gene’.

In a later period that overlapped the periods of phenotyping of theprevious pedigrees, another 861BC1 pedigree, designated ‘861BC1j’, wasgenotyped for two markers. The first HRM-marker was located in aA20/AN1-like zinc finger family protein gene, shortly ‘A20/AN1-like’(primers CM45/CM46, see Table 33), which replaced marker AO022, and thesecond marker was Asp_80 (CK63/CK64, Table 3 EXAMPLE 1)

At the time progeny 861BC1j was evaluated for berry set it was largelyunknown that some pedigrees may show a genetically determined, ratherthan an environmentally controlled, variable number of berry set andfruit set for those plants was roughly scored as capable or incapable offruit set, rather than assessed quantitatively.

A number of 142 plants was found that were ‘non-recombinant’ for theK1036 hermaphrodite grand-parental allele for markers ‘A20/AN1-like’ andAsp_80 flanking the GDS locus. Of those 142 plants; 118 produced berriesand 24 plants did not set fruit. A number of 135 plants was found thatwas ‘non-recombinant’ and had the DH02/504 male grand-parental allelefor markers A20/AN1-like and Asp_80 flanking the GDS locus, all of whichwere male and produced no berries. Six marker recombinants showed aphenotype that was expected based on their typed GDS allele.

The 24 plants that, despite of their alleles, which were of K1036hermaphrodite grand parental origin, did not produce berries, were keptfor phenotyping in the next season together with eight hermaphrodite‘control plants’ and eleven male ‘control plants’ (these control plantsshowed phenotypes consistent with their marker alleles A20/AN1-like andAsp_80 originating from hermaphrodite K1036 and male DH02/504grand-parents, respectively) together with three plants that had notbeen phenotyped before, one plant that had recombination event betweenA20/AN 1-like and the GUS gene and four that showed a recombinationevent between the GDS gene and Asp_80.

In this next evaluation, the number of berries as well as flowermorphology were determined more carefully. The eleven male controlplants again produced no berries at all and they had ill-developedstyles (score 1). The plants expected to be hermaphrodite based on theirgrand parental alleles of markers linked to the GUS locus showedvariability in fruit set and flower morphology. Of 36 plants that hadthe K1036 hermaphrodite grand-parental GDS allele, six plants producedover 100 berries, six produced 25-65 berries, seven produced 1-18berries and the remainder produced no berries at all.

This again showed that the loss of female suppression of the K1036 grandparental GDS allele was incomplete in this pedigree.

To find out whether the GDS allele originating from K1036 could be anepi-allele, as has been found for sex-determination in melon (Martin etal., 2009), it was decided to obtain bi-sulfite sequencing data forhermaphrodite K1036, the reference genome of male DH00/086 and for line9 male which has the same GDS haplotype as K1036 (because of sharedSNPs). Below the materials and methods are described that were used toobtain the data:

Libraries

Illumina sequencing libraries were prepared from bisulfite convertedDNA. Following bisulfite conversion unmethylated cytosines wereconverted to uracil whereas 5-methyl-cytosines remained intact.Following PCR-amplification the converted nucleotides yielded a thyminewhereas the non-converted nucleotides remained as cytosines.

For each library, 2 μg of total DNA were sonicated to ˜550nt using aCovaris-S2. End Repair was performed using the End-It Kit (Epicentre)according to manufacturer's instructions. The reaction was cleaned using0.8× AmpureXP beads. A-tailing was performed using Klenow (3′ to 5′ exominus, NEB) and incubated at 37° C. for 30 minutes. The reaction wasagain cleaned using 0.8× AmpureXP beads. NextFlex sequencing adapterswere ligated onto each A-tailed fragment using T4 DNA ligase (NEB) andincubated at 16 C overnight. The ligation reaction was cleaned twiceusing 1× AmpureXP. Bisulfite conversion was performed using theMethylCode kit (Life Technologies) according to manufacturer'sinstructions. Bisulfite-treated DNA was amplified with Kapa Uracil+2×Readymix according to the following protocol: 2 min at 95° C., 30 sec at98° C., followed by 4 cycles of [15 sec at 98° C., 30 sec at 60° C. and4 min at 72° C.] ended by 10 min 72° C.

The amplified bisulfite libraries were again cleaned using 1× AmpureXPand sequenced with paired-end 150nt reads on an Illumina NextSeq500.

Bioinformatics

Paired end Illumina reads were mapped to the Asparagus 2.0 referencegenome (source) using BWA-meth (Pedersen, 2014) with bwa-mem (Li, 2013)version 0.10 using the following command line(/usr/local/bin/bwameth.py—reference . . ./Genome/02.assembly_result/V2.0/Asparagus.V2.0.genome.fa-t10—calmd-pDH0086 DH0086_bisulfite_1.fq.gz DH0086 bisulfite_2.fq.gz). bwa-methcreates two computationally converted reference sequences, one for theforward or Watson strand in which all cytosines are converted tothymines and one for the Crick or reverse strand, for which all guaninesare converted to adenines. Read pairs were mapped to bothcomputationally converted genomes, when a pair was mapped to the Watsonor Crick strand with a mapping score higher than 40 the pair wasretained. When a pair matched both the Watson and the Crick strand onlythe highest scoring pair was retained. A custom read group tag in theresulting BAM alignment file “YD:Z:f” identified read pairs mapped tothe Watson strand whereas “YD:Z:r” identified reads mapped to the Crickstrand. Based on these tags, reads were split into Watson mapping andCrick mapping pairs using the following bash command: “samtools view-hall.bam |tee>(grep “{circumflex over ( )}@\|YD:Z:f”C|samtoolsview—Shb→Watson.bam)|grep “{circumflex over ( )}@\|YD:Z:r”|samtoolsview—Shb→Crick.bam”. Samtools (http://www.htslib.org/download) version1.2 was used.

Following mapping, a custom python script was created that iterated overall nucleotides in the genome. Creating such a (python) script can beachieved by any competent bioinformatician familiar with the art. Forall cytosines on both the Watson and the Crick strand the correctcontext, CG, CHG or CHH (where H is C, A or T) was determined. Anucleotide is considered to be in CG context even if the base followingthis dinucleotide pair is also a G, so the first nucleotide in thesequence “CGG” is considered to be in CG context on the Watson strand.The cytosine opposite to the G on the second position, which resides onthe Crick strand, is also considered to be in CG context, as the 3′downstream nucleotide on the same strand here is a G. Similarly, thecytosine opposite to the third G is considered to be in CHG context, asthe first 3′ downstream nucleotide on the Crick strand is a C, whereasthe second 3′ downstream nucleotide on that strand is a G. Methylationlevels were determined for the Watson and Crick strand separately bycounting the number of unconverted versus total nucleotides. On theWatson strand, converted nucleotides are represented by thymines (T)with a reference nucleotide C whereas on the Crick strand convertednucleotides are represented as adenines (A) with a reference nucleotideG. Using samtools (version 1.2) pileup the per-position conversion ratewas calculated for cytosines on both Watson and Crick strandsimultaneously. Methylation was only called for position for which nonucleotide polymorphism was evident. Methylation polymorphisms can bedistinguished from nucleotide polymorphisms (SNPs) because in case ofthe latter both Watson and Crick strand show evidence of a polymorphismwhereas a methylation polymorphism is only present on either the Watsonor the Crick strand. This is due to the fact that bisulfite conversiononly affects cytosines, leaving the guanine on the opposite strandintact. Given sufficient read mapping coverage on both strandsmethylation polymorphisms can thus be distinguished reliably fromnucleotide polymorphisms.

Results

The M locus linked gene with the name “Aof030575.3” (indicated by thecoding region of SEQ ID NO: 1 (see EXAMPLE 1) located on Scaffold_905Asparagus Version 2.0 reference genome having a DUF247 domain showedstriking differences in CHG methylation between K1036 and DH00/086 in a1434 base pair region from position 49.815 to 51.249. A total of 113nucleotides in CHG context are present in this region. For K1036, theaverage methylation level was 0.73 whereas for DH00/086 the averagemethylation level was 0.03. A student T-test performed in MicrosoftExcel v15.13.1 (150807) assuming unequal variance with a zero hypothesisof no difference between the average methylation level between K1036 andDH00/086 is rejected based on P(T<=t)=9.03E-61. The difference in CHGmethylation between K1036 an DH00/086 for scaffold_905 Genome Version2.0 between bases 49.815 to 51.249 (corresponding to positions309757-308323 in Scaffold_905 of FIG. 13) was highly significant. Theresult of the analysis is shown in FIG. 15.

As the bi-sulfite data revealed a markedly high CHG methylation in theK1036 GDS allele, it was decided to obtain bi-sulfite sequencing datafrom four 861BC1 siblings. All of these four siblings had the GDS allelefrom grand-parent K1036. However, two were highly hermaphrodite (n>100berries) whereas two other siblings were virtually incapable ofproducing berries. The hypothesis was that, if methylation plays a rolein repressing the female suppressor gene, it would be expected thatplants which have the GDS allele of hermaphrodite grandparent K1036 andremain highly methylated would be hermaphrodite whereas plants that forsome reason have (partly) lost this methylation would be ‘de-repressed’thus have an activated female suppressor and become less hermaphrodite,if not strictly male. The plants that were hypothesized to change theirphenotype from highly hermaphrodite into poorly hermaphrodite or evenmale because of their loss of methylation were designated ‘revertants’

Sanger reads from bi-sulfite treated genomic DNA were obtained by PCRusing primers that allowed amplification of the bi-sulfite treatedtemplate. Towards this end, the GDS gene sequence was imported inBisulfite Primer Seeker 12S; to make an in silico conversion of thesequence. Subsequently, this sequence was imported in Primer3(Untergrasser, 2012) to design and select primers. The visualrepresentation in J-Browse of the Watson and Crick sequence reads ofbi-sulfite treated DH00/086 and K1036 template allowed for a carefulselection of a relatively small target (100.300nt) that includeddifferential methylation (or single methylation polymorphism; SMP's).Primers were selected in such a way that these did not anneal to SMP'sas this would create mismatching in differentially methylated DNAtargets.

The primers designed were CS77 and CS78 (see Table 33) that allowed foramplification of a 256nt fragment using bi-sulfite treated template DNA.PCR was performed using Kapa Uracil Plus as polymerase (purchased fromSopachem, Ochten, The Netherlands) according to the manufacturersprotocol applied on bi-sulfite treated CTAB isolated DNA (Doyle andDoyle, 1990) using the EZ DNA Methylation-Lightning Th Kit (ZymogenIrvine, Calif. 92614, U.S.A). PCR fragments were Sanger sequenced byBaseClear, Leiden, The Netherlands. Sequence alignment in Geneious(Biomatters, Auckland, New Zealand) provided a ‘high quality sequencepart’, which was:

(SEQ ID NO: 113) TTTGGATGAAGAATGATGATGAGTTCCTAAAAATCATGATTTACGATGGGGCTTTCATGATTGAAATCATGATAGCGACCGTTGAACCATATGAGCGCACACCTTCTAGCTATCATGCCAAGGACCCAATATTCAAGAAGCCATACTTGGTCGAAGATCTTCGTGTAGATATGCTCAGGTTGGATAATCAAATTCCAATGAAGGTCCTGGAGATATTGTCTAAATTCTGCAAGAACAAGGTAAGGAATGT TAATGAAA

In this high quality sequence part, SMPs that stand out as ‘double peakDT SNPs’ were found at eleven positions, respectively: 79, 88, 103, 119,127, 142, 176, 196, 207, 220, and 227 whereas at many other positions(n=26) the bisulfite C to T conversion has been complete thus showing nodouble peaks but only thymines for the four samples, which wasindicative of successful bi-sulfite treatment for all samples analyzed.

To quantify the relative amount of cytosines as their relative peak plotheight within the mixed C vs T peak plots of those SMPs, the programMutation Surveyor 5.0 (Softgenetics, State College, Pasadena, U.S.A.)was used. The abi files were imported as ‘Sample Files’ and the highquality sequence FASTA file was imported as the required ‘Genbanksequence file’ in the Open File menu of Mutation Surveyor. Settings wereadjusted in the Process→Settings→Others menu in which the Methylationoption was checked. In the Set by User menu, only the CG>TG option wasunchecked, followed by pressing ‘Run’ in the Process dropdown menu andpressing the Mutation Quantifier button in the toolbar. The quantifiedmutations (SMPs) then appeared in a spread sheet from which thepercentage of cytosines in the particular SMPs were taken and summarizedin Table 34.

Clearly, the double C versus T peaks revealed single methylationpolymorphism or ‘SMPs’ in which the peak height of the cytosines washigher in the two hermaphrodite samples compared to cytosine peak heightobserved for the revertants. This means that the methylation was moreprominent in the two samples that were highly hermaphrodite, compared tothe ‘revertants’, which was a quantitative rather than an ‘all ornothing’ difference.

Because bi-sulfite sequencing is technically difficult as it breaks downthe target DNA, other methods were applied to quantify differentialmethylation in the GDS gene.

Towards this end, the sequence was inspected for SMPs that overlappedwith recognition sites of methylation sensitive/impaired restrictionenzymes.

By this procedure two assays were designed. In a first assay a fragmentis amplified using the primers CN67 and CP32 (Table 3 EXAMPLE 1) tocover a 353nt genomic region that includes two recognition sites of themethylation sensitive restriction enzyme EcoRII (CCWGG: targeting CmCTGGon the plus strand and CmCAGG on the minus strand, where mC is thetarget SMP) at positons 82.86 and 148-152 relative to the CN67 5′ primeend. In a second assay a fragment was amplified using the primers CP35and CN82 (Table 3, EXAMPLE 1) to cover a genomic region that includes asingle recognition site for the enzyme GsuI (targeting CTCmCAG andmCTGGAG on the plus and minus strand, respectively, where mC is thetarget SMP) at position 184-189 relative to the CP35 5′ prime-end.

Forty nano-grams of genomic template DNA, isolated using Sbeadex® miniplant kit (LGC Genomics GmbH, Berlin, Germany) on a KingFisher 96instrument (Thermo-Scientific, Breda, The Netherlands), was subjected toa four hour digestion individually using 2 units of EcoRII and GsuI(Life-Technologies) in 1× standard buffer in a 15 μl volume. Control DNAcomprised a similar incubation apart from that the enzyme was replacedby MQ water. Subsequently, 2 μl of this enzyme and non-enzyme treatedtemplate DNA was individually used in a 10 μl PCR on a C1000 TouchThermal cycler covered by a CFX96 Real Time System (Bio-Rad, Veenendaal,The Netherlands) programmed for 98° C. 1 min, 40 cycles of [98° C.: 10sec, 62° C.: 5 sec and 72° C.: 10 sec] using PhireII (Life technologies)and LC green Biofire defense, Salt Lake City, U.S.A)

TABLE 34 The percentage of cytosines (C + T = 100%) at eleven SMPs inSanger sequences reads from a 258 nt PCR fragment obtained frombisulphite treated genomic DNA template. Templates are 303 and 580obtained from two strong hermaphrodites (producing over 100 berries perplant) and 600 and 606 obtained from two ‘revertants’ of which oneproduced only a single berry and one does not produce berries at all,despite of their K1036 grand-parental DUF247 allele. For the forwardreads some SMPs could not be called because lack of (reliable)information; shown as ‘nd’. At 26 other positions the C to T conversionwas complete for all samples (results not shown). Note that thepercentage cytosines retained in both reads, which is indicative ofmethylation, is much higher for the hermaphrodites compared to therevertants. This suggests that the apparent loss of methylationreactivates the female suppressor gene which results in lower fruit setof the revertants. SMP number (1-11) and their fragment position 1 2 3 45 6 7 8 9 10 11 read Plant ID 79 88 103 119 127 142 176 196 207 220 227herma fw1_1 303 56.10% 34.78% 16.37% 48.66% 41.02% 33.81% 91.71% nd ndnd nd herma fw1_2 303 59.72% 47.69% 19.58% 46.75% 39.81% 36.86% 94.06%nd nd nd nd herma fw 580 69.03% 43.99% 20.36% 56.15% 43.26% 37.89%94.30% nd nd nd nd revertant fw 600 24.34% 16.38% 6.97% 20.31% 22.32%10.52% 35.80% nd nd nd nd revertant fw 606 34.12% 17.83% 6.58% 28.08%21.65% 13.19% nd nd nd nd nd herma rev 303 50.22% 22.32% 18.86% 45.45%34.18% 22.73% 80.73% 20.02% 48.68% 12.13% 96.08% herma rev 580 73.96%36.65% 26.51% 58.04% 42.66% 35.43% 90.47% 29.37% 95.74% 21.02% 96.20%revertant rev 600 19.72% 6.27% 0.00% 10.87% 12.78% 5.35% 23.78%  5.19%19.68%  0.00% 85.89% revertant rev 606 32.69% 8.92% 0.00% 18.97% 19.31%9.05% 75.91%  8.41% 69.42%  0.00% 64.70% The percentage of cytosines(C + T = 100%) at eleven SMPs in Sanger sequences reads from a 258 ntPCR fragment obtained from bisulphite treated genomic DNA template.Templates are 303 and 580 obtained from two strong hermaphrodites(producing over 100 berries per plant) and 600 and 606 obtained from two‘revertants’ of which one produced only a single berry and one does notproduce berries at all, despite of their K1036 grand-parental DUF247allele. For the forward reads some SMPs could not be called because lackof (reliable) information; shown as ‘nd’. At 26 other positions the C toT conversion was complete for all samples (results not shown). Note thatthe percentage cytosines retained in both reads, which is indicative ofmethylation, is much higher for the hermaphrodites compared to therevertants. This suggests that the apperant loss of methylationreactivates the female suppressor gene which results in lower fruitsetof the revertants.

TABLE 35 The CQ values are provided for several backcross individualstyped for gender; segregating for hermaphroditism (andromonoecy) andmale phenotypes. Note that in the 861BC1d population individuals (lowerTable part), the CQ value is low for plants that have received thegrandparental allele from K1036 and that these plants are hermaphrodite,where for plants that received a DH02/504 allele an oppositerelationship has been found; high CQ values and a male phenotype.. Forplants from other populations 861BC1, a, b, c, e, (top of the Table 35)the CQ values can be much lower for plants having a K1036 alelle,notably those which were found to be male rather than hermaphrodite,these are typed as revertants Gsul replicate 1 Gsul replicate 2 EcoRIIreplicate 1 EcoRII replicate 2 Gender Population ID DUF247 allele origindelta CQ delta CQ delta CQ delta CQ revertant 861BC1a 408877 K1036 5.95.77 2.6 1.61 revertant 861BC1b 409019 K1036 7.1 5.15 3.7 1.9 revertant861BC1b 409022 K1036 4.5 2.97 4.7 2.37 revertant 861BC1c 409274 K10363.8 3.08 3.2 2.71 hermaphrodite 861BC1b 409151 K1036 −0.2 0.33 0 0.23hermaphrodite 861BC1e 409455 K1036 0.9 −0.66 1.2 −0.11 hermaphrodite861BC1e 409449 K1036 0.3 0.08 −0.4 0.01 hermaphrodite 861BC1e 409453K1036 −0.1 0.02 0.4 0.48 Gsul replicate 1 Gsul replicate 2 GenderPopulation ID DUF247 allele origin DNA of November 2012 DNA of April2013 male 861BC1d 377131 DH02/504 1.32 2.28 — — male 861BC1d 377139DH02/504 1.60 2.70 — — male 861BC1d 377183 DH02/504 1.28 — — — male861BC1d 377184 DH02/504 3.23 2.08 — — male 861BC1d 377104 DH02/504 2.653.91 — — male 861BC1d 377097 DH02/504 2.85 1.02 — — male 861BC1d 377119DH02/504 1.08 2.74 — — male 861BC1d 377109 DH02/504 3.45 1.91 — — male861BC1d 377127 DH02/504 4.76 4.39 — — male 861BC1d 377163 DH02/504 2.684.89 — — male 861BC1d 377185 DH02/504 2.19 5.01 — — hermaphrodite861BC1d 377182 K1036 −0.49 −0.02 — — hermaphrodite 861BC1d 377162 K1036−0.18 −0.38 — — hermaphrodite 861BC1d 377134 K1036 0.27 −0.32 — —hermaphrodite 861BC1d 377180 K1036 0.23 −0.04 — — hermaphrodite 861BC1d377110 K1036 −0.10 −0.04 — — hermaphrodite 861BC1d 377142 K1036 −0.060.57 — — hermaphrodite 861BC1d 377122 K1036 0.01 0.22 — — hermaphrodite861BC1d 377124 K1036 0.72 0.43 — — hermaphrodite 861BC1d 377152 K1036−0.68 0.58 — — hermaphrodite 861BC1d 377096 K1036 −0.34 — — —hermaphrodite 861BC1d 377102 K1036 −0.54 — — — CQ values were determinedby a cut off threshold value of 500 CFU.

The CQ value difference (delta CQ), which is the CQ value obtained fromdigested template DNA minus the CQ value of non-digested template DNA,was used as a measure of DNA methylation.

The result of this pilot is shown in Table 35. Results show thathermaphrodite plants had a delta CQ value of about zero, indicative ofhigh methylation (as the enzyme is not able to cut the template offeredfor PCR), whereas the revertants had a delta CQ value that is largerthan zero ranging 1.9-7.1, indicative for poor methylation in the GDSgene region, targeted by this method. For population 861BC1d it showedthat male plants which have the DH02/504 grand parental GDS allele havedelta CQ values larger than zero, whereas the hermaphrodites which havethe K1036 grand parental GDS allele had delta CQ values approachingzero.

This shows that this method can be used to monitor a male plant for itshermaphrodite tendency, thus its capability to produce berries. Theskilled person will recognize that the method of methylation sensitiverestriction enzyme digestion, followed by Q-PCR is a rough method andnot perfect. For instance results presented in Table 35 reveal that onehermaphrodite (ID: 409455) showed a delta CQ of 1.2 (rather than aboutzero) for EcoRII replicate 1. The skilled person will understand that touse this method optimally, several replications and the use of moretargets, preferably using several methylation sensitive restrictionenzymes is preferred. In conclusion, similar to what has been observedin the bi-sulfite sequencing experiment, in the Q-PCR experimentdifferential DNA methylation has been detected as inferred from thedifference in CQ of methylation impaired restriction enzyme treatedtemplate DNA relative to the non-treated template DNA used for PCR. Forthe revertants and also for the males, the low methylation stood out asa high relative difference in the CQ values obtained from methylationimpaired restriction enzyme treated template DNA relative to thenon-treated template DNA used for PCR. This differential methylationclearly and stably segregated in backcross population 861Bc1d. Themethylation of the GDS grandparental allele of K1036 was shown to beunstable in other pedigrees consistent with the hermaphrodite phenotype,which was also unstable. Micro-satellite marker analysis, using morethan five hyper-variable loci, has demonstrated that those unstableplants or revertants were plants that truly belonged to those pedigrees(results not shown).

There is an increasing number of scientific papers that report on themethylation of genes and genomes and the inheritance of epi-alleles(e.g. Ji et al., 2015; Greaves et al., 2014, Zhang et al., 2013). Inplants DNA methylation is separated in three distinct contexts; CG, CHG,and CHH (where H=A, T or C). Regions of the genome methylated in allthree contexts often lead to silencing in the targeted region and insome cases neighboring regions (see reference in Ji et al., 2015). Manyof the silenced genes have a lower expression because of promotermethylation spreading from repeat sequences (or duplications) into genes(cmWIP1, booster1, BSN, FOLT1; see Ji et al 2015). There are someexamples in which methylation of exons, rather than in the promoter,results into a lower expression. One of the earliest examples is foundin the so called clark kent (clk) alleles of the SUPERMAN gene inArabidopsis. Superman is a gene which results into a higher number ofanthers when knocked-out out by gene mutations for which allelic formswere found that provide the same phenotype but revealed no nucleic aciddifferences from the wild type. Bisulfite sequencing however revealedfor those (elk) phenotypes that there was no cytosine methylation inwild type or in a sup ‘nonsense’ allele (sup-1) whereas extensivemethylation in all contexts was found in the elk alleles covering thestart of transcription and most of the transcribed region.Interestingly, also revertants and stronger and weaker elk alleles wereobserved that were related to DNA methylation. The phenotype reversionis correlated with both the restoration with the wild type RNAexpression and a decrease of cytosine methylation of the SUPERMAN geneDNA.

The skilled person will understand that the methylation observed for theasparagus GDS locus, and the phenomenon of revertants related to reducedmethylation as was demonstrated in the present document, mirrors thesituation found for SUPERMAN. For technical reasons combined with thelow wild type expression of the GDS gene it appeared impossible tounequivocally determine whether the GDS gene methylation results into alower expression or alternative sequencing. The skilled person willunderstand such a relationship is very likely and that gene capturingtechniques followed by RNAseq studies, sampling specific tissues ordevelopmental stages will likely confirm the relationship between thehermaphrodite phenotype and the methylation of the GDS gene and thelowered expression levels or splicing.

This present example reports on epigenetic control of hermaphroditism inlot K1036 and its derived progenies. It discloses that methylation ofthe GDS gene provides a method to obtain a hermaphrodite plant. Thepresent example also demonstrates that methods that allow the detectionof methylation, such as but not limited to bisulfite sequencing (partsof) the GDS gene or the use of restriction enzymes impaired bymethylation that target the GDS gene, can be used in diagnostics topredict whether a plant has a tendency to become or to stayhermaphrodite.

The skilled person will recognize that there are many methods that allowfor the detection of DNA methylation such as, but not limited to methodsreviewed by Shen & Waterland, 2007 and that any such method can be usedin the present invention.

The skilled person will also recognize that influencing DNA methylationof the GDS either by increasing or reducing it, will result in changesin either gene expression and/or splicing that will reduce or increasefemale suppression. Methylation in the present example is confined tothe transcribed region but the skilled person will also understand thatmethylation of the promotor or other cis acting elements near the genemay result into reduced expression or alternative splicing and thus mayresult into reduced female suppression. Methylation in all context couldhe established by virus induced gene silencing and a part of thismethylation may persist even after the virus has been eliminated (e.g.see Dalakouras et al., 2012).

Other methods to establish gene methylation are proposed by Zhang &Hsieh (2013) who state that crop improvement via locus specificepigenetic manipulation has become increasingly feasible with TALE orCRISPR-based genome editing techniques.

Recently, targeted methylation has been achieved to reduce theexpression of a gene that is expressed in many forms of human cancers(Nunna et al., 2014), by targeted methylation of the gene its promoter.These authors used an engineered Zinc Finger that specifically binds toa gene promoter that has been fused to the catalytic domain of a DNAmethyl transferase. The skilled person will understand that anytechnique that provides a catalytic moiety which delivers a silencingsignal together with a targeting part, which ensures the specificbinding of the catalytic moiety to a defined genomic target may allowtargeted methylation. Such techniques may be developed in near future.Rather than Dnmt3a methyl transferase as has been used by Nunna et al.,a methyltransferase is preferred that enhances the CHG methylation ofthe coding region of the DUF247 gene. A similar hypermethylation effectmay be achieved by targeted histon modification. Examples of genesinvolved in non-CG methylation are reviewed in Stroud et al (2014). Thepresent example teaches that methylation in all contexts but notably CHGmethylation of the exons and intron of the DUF247 gene will results infeminization.

Example 4

A Histidine to Glutamine Mutation in the Second Predicted Exon of theGDS Gene

Cultivar K5756 is an all-male hybrid cultivar which is a cross between aclonal female plant; 169F1-85V and a doubled haploid male plant;DH05/128. The latter doubled haploid was selected as parental plantbecause it, among other criteria, was not capable of producing berries

First year plants of this hybrid were first raised at a nursery farmafter which the crowns were replanted in a hybrid evaluation field.

There was a small chance that a crown could be divided in two crownswhen crown were bagged prior to transplanting.

Hybrid K5756 was trialed in four replicate plots of twenty plants each.

When evaluated, two different plants in the same plot were full ofberries whereas all other individuals of this hybrid in any of the fourplots bore no berries at all and those berries comprised viable seeds Atthe moment of inspection late in the season some blown flowers werestill present on the two plants bearing berries which showed theremnants of anthers and large petals which confirmed their apparentpistillate and staminate, thus truly hermaphrodite nature. Berriesharvested from the two plants provided 1016 seeds in total. Fern wastaken from the two hermaphrodites and a control plant of the samehybrid. Template DNA obtained from the two hermaphrodites was Sangersequenced in both forward and reverse direction using primerscombinations CN82/CN67, CN59/CN70, CN69/CN81. Sequence reads obtainedfrom both primer pairs CN59/CN70, CN69/CN81 disclosed a similar cytosineto arginine transversion. This transversion disrupts the second of intotal three HphI restriction sites (in this case 5-{circumflex over( )}(N)7TCACC-3) present in the CN69/CN70 PCR fragment, and thus thatfragment could be used in diagnostics. This type of diagnostics wasperformed on the two hermaphrodites and a male control sample taken fromthe field. This analysis, commonly referred to as CAPS marker analysis,confirmed that the particular transversion was unique for the twohermaphrodite plants. Microsatellite analysis using seven proprietaryhypervariable loci showed that the two hermaphrodites had an identicalgenotype which differed from the control sample.

However, both the alleles observed in the hermaphrodite and the maleplants confirmed that these belonged to the same hybrid. In conclusion,a mutation was found in the M-linked GDS gene in two hermaphroditeclonal copies in all male hybrid 5756. This clone provided the onlyhermaphrodite specimens found in this hybrid. The particular mutationchanges the cytosine (at position 684 of SEQ ID NO:1) at the third codonposition of a histidine (H) into a adenine, providing a codon for aglutamine (Q); thus CAC>CAA.

Example 5

A Mutation Changing a Proline to Threonine in the Second Predicted Exonof the GDS Domain Containing Female Suppressor Gene Creates anHermaphrodite

Cultivar K4381 is an all-male hybrid cultivar which is a cross betweenfemale doubled haploid DH366/1 and male doubled haploid DH02/047, eachof which were obtained by anther culture. D1102/047 was selected amongother criteria, as parental plant of this hybrid because it produced noberries. For over 190 genetically different hybrids made by DH02/047tested there were no reports of fruit set in our breeding databaseIndividuals of such doubled haploid× doubled haploid hybrids as K4381,are genetically identical.

The cultivar K4381 was grown in a 4 times 20 plants (thus n=80plants)field trial. Among these eighty plants, a single plant wasidentified that was fully hermaphrodite. This single plant producedhundreds of berries, comprising viable seeds whereas all otherindividuals produced no berries at al. This hermaphrodite off-type plantwas analyzed for microsatellite markers which showed that it was fullyidentical to a reference individual of this particular K4381 cultivar(results not shown). In conclusion, this hermaphrodite individual wasnot the result of a genetic impurity within this trial. To find outwhether a mutation in the GDS gene generated the K4381 hermaphroditeplant, sequences were obtained for this K4381 hermaphrodite plant usingthe primer pairs CN82/CN67, CN59/CN70, CN69/CN81. These primers span thefirst predicted exon, the predicted intron 1 and the second predictedexon of the GDS gene.

Compared to the reference genome, a polymorphism was found that wasalready previously identified. This polymorphism comprises a stretch ofseven thymine's rather than six, close to the predicted intron 1acceptor site found at scaffold 905 (genome version 2.0) position50,941-50,946, which is also found in similar haplotypes such as insuper-male 12_25, hermaphrodite 5375, all male hybrid K323 andhermaphrodite mutant K323-G033. More importantly, a single nucleotidepolymorphism (SNP) was found for which hermaphrodite individual K4381 isunique compared all haplotypes known and sequenced so far. This SNP is acytosine to an adenine change in the first predicted exon, correspondingto position 166 of SEQ ID NO:1. which leads to a proline into athreonine amino-acid change at position 56 of SEQ ID NO:2

This particular mutation is a non-conservative substitution as anon-polar amino-acid is changed for a polar amino-acid. The presentexample teaches that this particular amino-acid change in the GDS geneapparently is sufficient to change a male into an hermaphrodite plantwhich produces much more berries than its male ancestor.

Example 6

An Asparagus Homologs of Defective in Tapetal Development and Function 1is the Male Activator Gene

To isolate the male promoter gene, the M-locus region was furtherinvestigated by applying BioNano Genome Mapping (Bionano Genomics). Bythis approach, DNA sequence genome scaffold (including scaffolds taggedby sex linked markers) were aligned to BioNano contigs, and one contig,likely spanned the M-locus. New genome sequencing scaffolds wereidentified and on one of those scaffolds in a part of the genome wherefemale reads do not map to the male reference genome, a candidate genehomologous to As-TDF1 was identified.

The hemizygous presence of TDF1 in males; the phenotype of its deletionmutants and a study of expression and genomic read mapping in ofAsparagus genes homologous to member genes, expected to act in thepathway downstream AS-TDF indicate that AS-TDF1 is the male stimulatorgene.

High Molecular Weight genomic DNA of the Asparagus officinalis genotypesDH00/086 and DH00/094 was isolated. DH00/086 is the supermale used bythe Leebens-Mack laboratory University of Georgia at Athens to create areference genome of asparagus. DH00/094 is a female doubled haploidobtained by tissue culture from the same hybrid from which the doublehybrid male DH00/86 originates (Limgroup BV, Horst, The Netherlands) Forthis, fresh leaves were washed in 10 mL of TEN buffer (10 mM Tris, 10 mMEDTA, 100 mM NaCl, pH7.5) and fixed in freshly prepared TEN/2%formaldehyde solution. The leaves were chopped in very small pieces andincubated in 15 mL Isolation Buffer (IB: 15 mM Tris, 10 mM EDTA, 130 mMKCl, 20 mM NaCl, 8% (m/V) PVP10, pH19.4) containing+0.1% Triton X-100 torelease the nuclei. The nuclei were purified by density gradientcentrifugation on 20 mL of 75% Percoll in IB/0.1% Triton X-100 for 20min at 2000 RPM. The resulting stabilized homogenate was embedded in anagarose matrix by gentle mixing with IB/1.5% Low Melting Point agaroseat 60° C. followed by poring the mixture in a precooled agarose plugmold cast (Bio-Rad, Hercules Calif., USA)) on ice for 10 min. The 220μl, plugs were collected in lysis buffer (1% sarkosyl, 0.25 M EDTA pH8.0 and 0.2 mg/ml proteinase K) at 50° C. for one day with one change oflysis buffer. After extensive washing in TE buffer the HMW DNA wasrecovered by gentle melting at 60° C. and GELase™ (Epicentre, Madison,Wis., USA) treatment using 3 units of Gelase™ per plug for 10-20 min.The High Molecular Weight (HMW) DNA was further cleaned by drop dialysisprior to quantitation on CHEF electrophoresis (CHEF-DRII system,Bio-Rad, Hercules, Calif., USA). On average, 3-4 μg HMW DNA was obtainedper plug.

The HMW DNA was processed in-house at BioNano Genomics Laboratories(BioNano Genomics, Inc., San Diego, Calif., USA) creating Genome Mapsi.e. long range Physical Maps (reviewed by Brown, 2002) of the Asparagusmale and female genomes using their proprietary Irys Technologypipeline. The Irys Technology involves labelling HMW DNA withfluorescent dyes (IrysPrep®), movement of single molecules innanochannels (IrysChip®), scanning of the molecular position of the dyesby a CCD camera (Irys Instrument) and de novo assembly and visualizationof Genome Map contigs (Irysview Software®, Shelton et al., 2015).

Briefly, 8 μg of HMW DNA was labelled according to protocols in theIrysPrep® method. The HMW DNA was nicked with the nicking endonucleaseNt.BspQI at GCTCTTCN/N positions (New England Biolabs, NEB, Ipswich,Mass., USA). Nicked DNA was labelled with Alexa546-(Thermo FisherScientific, Waltham, Mass., USA) and Taq polymerase (NEB). Afterlabelling, the DNA was ligated by adding dNTPs and T4 DNA ligase (NEB).The labelled DNA samples were pipetted onto individual IrysChip® in bothflow cells. The Irys Instrument controls the movement of DNA in the flowcells electrophoretic ally. Linearized molecules were imaged using greenlasers for Alexa546. A CCD camera, coupled with proprietaryauto-focusing mechanism and control software, rapidly scanned the chips.Next, the locations of labels (Alexa546) along each moleculeindividually were detected and analysed using the Irysview Software®package. Raw image data of labelled long DNA molecules are converted todigital representations of the motif-specific label pattern. First, theraw image data of labelled long DNA molecules were converted to digitalrepresentations of the motif-specific label pattern. Next,single-molecule Nt.BspQI data were clustered by scoring all moleculemaps for similarity to one another and clustering by the R-packageFastcluster (Daniel Mülliner, 2013). From the clusters, the labellocations were plotted. Finally, the data were assembled de novo usingIrysView® data analysis software to recreate a whole genome consensusmap of the original genomes of Asparagus officinalis genotypes DH00/086and DH00/094. For Asparagus officinalis genotype DH00/086 (Male) 88Gb(79X) of data was collected (molecules>150 kb). The resulting BioNaonoGenomics consensus assembly size was 1.205Gb contained in 1364 contigs.The contig assembly of the data exhibited a contig N50 of 1.24 Mb. Thecontig database is referred to as BNG V1.0 and individual contigs asprefix <BNG>number.

The scaffolds Reference genome of Asparagus obtained by NGS (AGS V1.10)were linked to the BNG V1.0 contigs using the Irysview Software®package. First the AGS V1.10 was upgraded by aligning long sequencingreads obtained by PacBio RS II sequencing (Pacific Biosciences, CA, USA)to the AGS V1.10 scaffolds using an algorithm and associated softwaretool named BPJelly (English et al., 2012). PBJelly is a highly automatedpipeline that aligns long sequencing reads in fasta format to draftassembles. PBJelly fills or reduces captured gaps (N-stretches in AGSV1.10) to produce upgraded draft genomes. Briefly, High Molecular Weight(HMW) genomic DNA of the Asparagus officinalis genotypes DH00/086 andDH00/094 was isolated as described before and used as input for PacBioSMRTbell library preparation according to the manufacturer instructions(Pacific Biosciences, CA, USA). The prepared library was size selectedfor >20Kb fragments using the BluePippin System for targetedsize-selection of HMW DNA (Sage Science, MA, USA). The collectedfraction was sequenced within 2 SMARTcells on a PacBio RS II sequencerat the University of Florida Interdisciplinary Center for BiotechnologyResearch (ICBR, USA). Nearly 6.07Gb of long read sequencing data weregenerated corresponding to 4.6× coverage of the Asparagus officinalisgenome. FIG. 16 displays the observed length distribution of the PacBioexperiment. PBJelly was run at Beijing Genomics Institute (BGI,Shenzhen, China). The resulting Reference Genome is referred to asAsparagus Genome Scaffold V2.0 (AGS V2.0) and individual scaffolds asprefix <AsOf_V2.0_scaffold>number. The annotation metadata were storedas individual files in AGS V2.0 based relational databases.

The AGS V2.0 scaffolds larger than 20 Kb (5198 AGS V2.0 scaffoldsrepresenting 1,113 Mb) were used in mapping to the BNG V1.0 contigs bydetecting the recognition sequences of nicking endonuclease Nt.BspQI atGCTCTTCN/N positions in silico. The resulting physical maps of the AGSV2.0 scaffolds (Query_id) were aligned to the BNG V1.0 physical maps(Anchor_id) using the Irysview Software® package with standard settingsof stringency. This software creates Matches (Match_ids) of Anchor_idsand Query_ids. In total, 2725 AGS V2.0 scaffolds (52%) were aligned tothe BNG V1.0 contigs representing 875 Mb (79%). The resulting comparisonmap (cmap) was stored as

Asparagus.V2.0.genome.stable_BspQI_res29_to_20150505_asparagus_UGA_Assemble_Molecules.xmap and could be viewed using the Irysview Software® package byhighlighting the data in the Compared Maps mode. Within this environmentseveral aspects of the cmap could be visualized and a table of Matchesfor each individual Match_id listing corresponding Anchor_id,AnchorStart, AnchorEnd, Anchor size, Query_id, QueryStart, QueryEnd andOrientation of the Query_id with respect to the Anchor was included.Table 61 summarizes the results of the Compared map for Asparagusofficinalis genotype DH00/086 (Male) V2.0 scaffolds that were detectedusing genetic marker information, such as the HRM markers from Table 3,and physical information (BAC clones; results not shown) The firstcolumn shows the ASG V2.0 scaffolds used for comparison based on geneticmarker information in the third column (sex linked) and correspondingBioNano V1.0 contigs. A total of eight corresponding BioNano contigs(BNG7, BNG22, BNG28, BNG55, BNG438, BNG833, BNG1030 and BNG1138) weredetected and it was established by inspection of the nicking data of thelisted BNG contigs that there were no physical overlaps between thesecontigs. These data (Table 61) strongly suggest that all eight contigscluster on the chromosomal region covering the M-locus of Asparagusofficinalis. All eight contigs were inspected for the sequence contentof aligned AGS V2.0 scaffolds and their collinearity between their BNGV1.0 and AGS V2.0 cmaps.

BNG28 is 3.45 Mb in length and the cmap shows linearity for the GDScontaining AsOf_V2.0_scaffold905 as well as the sex-linkedAsOf_V2.0_scaffold206, AsOf_V2.0_scaffold945, AsOf_V2.0_scaffold1194,AsOf_V2.0_scaffold1204, AsOf_V2.0_scaffold1539 andAsOf_V2.0_scaffold2312 (FIG. 17, Table 3). The sex linkage of thesescaffolds has been previously demonstrated using molecular markers inpopulations segregating for gender (results not shown). Markers thathave been used to test the sex linkage of those scaffolds are listed inTable 3In addition, four scaffolds, AsOf_V2.0_scaffold436,AsOf_V2.0_scaffold2510, AsOL_V2.0_scaffold3294 andAsOf_V2.0_scaffold3779 matched BNG28 and were not identified before(labeled ‘new’ in the third column of Table 61), The cmap of BNG28 andthe 11 indicated AGS V2.0 scaffolds revealed the linear order of thescaffolds on BNG28, the orientation of the scaffolds and the chimericnature of five scaffolds. Chimeric nature is defined as the joining ofone or more sequence assemblies in scaffolds of Asparagus officinalisV1.10 and V2.0 that are not reflecting the original genomic DNA sequenceused in Next Generation Sequencing and Genome Assembly. As a result,AsOf_V2.0_scaffold206, AsOf_V2.0_scaffold436, AsOf_V2.0_scaffold945,AsOf_V2.0_scaffold1204 and AsOf_V2.0_scaffold2312 were found to bechimeric. This was confirmed by the presence (not MSY) or absence (MSY)of female reads of DH00/094 resequencing data in a JBrowse environment(JBrowse 1.1.16, Skinner et al., 2009, MSY refers to the male-specificregion of the Y chromosome, which is a term taken from human genetics(but also applied for dioecious plants such as papaya; see Yu et al2009) meant to clarify that the genome segment is male specific, whichmeans that reads obtained from a sequenced female will not show, thuslack, reads mapped to such a region of a male reference genome.

The seven remaining AGS V2.0 scaffolds in table 61 known to besex-linked that matched to a cmap other than BNG28 were also inspectedfor their chimeric nature and the positions of the genetic markersequences within these scaffolds. From the seven scaffolds,AsOL_V2.0_scaffold997 and AsOL_V2.0_scaffold1166 were found to bechimeric. The non-matching sequences of these scaffolds were extractedand used in a new mapping to the BNG V1.0 contigs essentially asdescribed for the AGS V2.0 scaffolds representing 1,113 Mb. As a result,AsOf_V2.0_scaffold997 Region=1 . . . 140,022 that did not match toBNG222 and containing a sex-linked marker (data not shown) mapped toBNG28 at positions 1,093,801 . . . 1,169,913 overlapping with thenon-colinear region of AsOf_V2.0_scaffold436. The non-matching sequenceof AsOf_V2.0_scaffold1166 aligns to BNG37.

All AGS V2.0 cmap regions that were strictly colinear with BNG28 wereeither extracted and used for AUGUSTUS Gene Prediction (Hoff et al.,2013) or manually inspected in JBrowse environment. The translatedannotations were used as Query in the alignment software BLASTP ProgramBlast2.3.0 using a database of the non-redundant protein sequences (nr)of Genbank CDS translations plus protein sequences in the databases PDB,Swissprot, PIR and PRF excluding environmental samples from WGS projects(ncbi.nlm.org updated October 2015 version 210). The sequences werelimited to the Viridiplantae [ORGN] including a filter for lowcomplexities. All other settings were default. The resulting BLASTscores were filtered (e-values<1E-40) and manually curated formis-annotations and checked read coverage of female DH00/094e inJ-Browse Next to the DUF247 gene model, proven to be involved in femalesuppression, now designated the GDS gene, two other gene models werefound that could be involved in flower developmental fate of maleness,femaleness and hermaphroditism: PREDICTED: LIPID TRANSFER PROTEIN1(LTP1) Gene Model At2G38540 in Arabidopsis thaliana onAsOf_V2.0_scaffold905 and PREDICTED: transcription factor MYB34 [Phoenixdactylifera] on the part of AsOf_V2.0_scaffold436 that is colinear withBNG28 Region=380,000 . . . 496,167. The LTP1 gene maps in the linearorder of BNG28˜280Kb distal to the DUF247 Gene Model, now designated theGDS gene and genetic mapping experiments using informative markersbetween these two Gene Models show that LTP1 is not fully sex-linked(Limseeds BV, Horst, The Netherlands). The MYB34-related Gene Model is˜600Kb proximal to the DUF247 Gene Model now designated the GDS gene.The MYB34-related Gene model was further investigated since severalstudies indicate that MYB-class transcription factors are key regulatorsin pathways involved in developmental processes and general stressresponses. MYB33, MYB35, MYB65 and MYB103 are transcription factorsacting in gene regulatory networks involved in later stages of stamendevelopment, more precise the stages described as tapetal development inearly microsporocyte development (Jun Zhu et al., 2008, Harkess et al.,2015, Ci-Feng Cai et al., 2015). The MYB34-related Gene Model wasinspected by Sanger sequencing using several gene-specific primers andone N-stretch could be filled using de novo assembly of the gap usingRNA-Seq data. One inverted repeat was discarded from the assembly. Thereconstructed MYB34-related Gene Model has three introns (FIG. 18) andcodes for a 276 AA Protein of 31 Kdal (FIG. 19). When re-used as Queryin BLASTP, using a database of all non-redundant Genbank CDStranslations, the SmartBlast option was used. The SmartBlast option inNCBI Blast environment returns a concise summary of the best matches inthe sequence database together with the two best matches fromwell-studied reference species, showing phylogenetic relationships basedon multiple sequence alignment, and conserved protein domains. UsingSmartBlast in standard settings the output was: protein DEFECTIVE INMERISTEM DEVELOPMENT AND FUNCTION 1 (thale cress), PREDICTED:myb-related protein 308 (chickpea), PREDICTED: transcription factorMYB35-like (soybean), PREDICTED: transcription factor MYB76 (Nelumbonucifera), PREDICTED: transcription factor MYB34 (date palm). TheArabidopsis thaliana DEFECTIVE IN MERISTEM DEVELOPMENT AND FUNCTION 1gene belongs to the MYB35-subclass of MYB-containing gene family and ischaracterized by two DNA-binding SANT Superfamily domains (also referredto as R2R3 sub-class). Binding is sequence dependent for repeats whichcontain the G/C rich motif [C2-3A (CA)1-6]. The domain is strictly foundin the Plant Kingdom as part of regulatory transcriptional repressorcomplexes where it binds DNA (reviewed in Jin and Martin, 1999). TheDEFECTIVE IN MERISTEM DEVELOPMENT AND FUNCTION 1 gene of Arabidopsisthaliana has been mapped-based cloned by using a single mutant line anda mapping population derived thereof (Jim Zhu. 2008) and was renamedDefective in Tapetal Development and Function 1 (ATH TDF1) describingits essential role in anther development and tapetal function formicrospore maturation in Arabidopsis thaliana. The Asparagus officinalisMYB34-like gene used as Query also belongs to the MYB35-class oftranscription factors and shares high sequence identities in the SANTSuperfamily domains with ATH TDF1. The MYB34-like Gene Model wastherefore renamed AsOf TDF1-like. The SANT Superfamily domain in AsOfTDF1-like is found twice at residues 16(H)-60 (Y) and 76(F)-K(151).Members of MYB35-related proteins are ˜300-350 amino acids whereas AsOfTDF1-like has 276 amino acids; the proteins have high identities in theN-terminal SANT Superfamily domain organization and sequence identitiesare lower towards the C-terminal end of the proteins. When the ATH TDF1protein sequence is taken as Query in AGS V2.0 database, the tBLASTNoutput has two significant hits: next to the AsOf_V2.0_scaffold436 alsoAsOf_V2.0_scaffold1220, the latter having less identity in the highlyconserved first SANT Superfamily domain (78% versus 52% see FIG. 20).

In order to find out whether male sterile plants thus lacking afunctional TDF1 gene could be obtained renewed irradiation experimentswere performed.

Seed lots of three different all-male hybrids, designated K1150, K323,and K1129 all of which originated from crosses between doubled haploids,thus which per seed lot would yield genetically similar individuals,were subjected to a dose of 300 gray (n=11.00 seeds) and 600 gray(n=13,000 seeds) irradiation from a Cobalt 60 source as has beenexplained in EXAMPLE 2.

The father plants of these hybrids were, among other criteria, selectedbecause these were virtually incapable of producing berries. K1129 hasonce before sporadically produced a few berries in one year in one of atotal of six trails and these plants have not been further investigated,K323 and K1150 never produced berries in multiple trials.

Plants raised from these seeds were grown in seedling trays from whichplants were finally transferred into an evaluation field near Trujillo(Peru). The particular hybrids were chosen because these have notendency to produce berries spontaneously as was established duringtheir previous evaluation, throughout the years. Any berry produced onplants therefore would thus be indicative of a mutation that caused thisability to produce berries. A number of 6,680 plants obtained from24,000 seeds that survived the irradiation treatment were inspected forfruit set after 10 months of plant growth, where after four months thefern was cut to obtain renewed flowering and/or fruit set that wasobserved 6-8 weeks later (November-December 2015) three times by ourlocal assistants. The majority of those plants originated from a 300gray dose as for the 600 gray dose only 1492 plants from 13,000irradiated seeds survived the treatment. Sixteen plants were found to becapable of producing berries from at least one of their branches. Thenumber of berries formed per plant varied from 1 to 174 berries.However, because plants were heavily infected by the citrus gall midgeProdiplosis longifila Gagné which had caused damage on the berries andcaused fruit abortion, the number of berries found on a plant could notbe interpreted as a quantitative measure of female fertility. Inconclusion: the presence of more than one berry was a qualitativeindication of female fertility. One of the 16 plants, capable ofproducing berries (K1150-600-1) had two female flowers. In a second stemflush photographs of both K1150-600-1 and K323-600A6 could be taken butnot of the third plant showing the deletion (K1150-300-12) that did notretained its growth after a fern cut, but from which F1 plants arecurrently growing in our greenhouse for further analysis.

Template DNA of the plants that were capable of berry production andsome DNA of non-berry producing male control plants was used in HighResolution Melting Curve analysis (essentially performed usingguidelines described in Gady et al., 2009) using primer pairs CP31/CP32,CP33/CP34, CP35/CP36, CP37/CP38, CP39CP40, CP41/CN72 targeting theDUF247 containing M locus linked female suppressor gene or GDS gene.These primers are listed in Table 3. Fragments were analyzed for meltingcurve differences that would be indicative of a mutation in the M-locuslinked Gynoecium Development Suppressor (GDS) gene. It appeared thatfragments could not be amplified or give rise to a melting curve shapethat looked very different compared to the wildtype melting curves forthree of the sixteen plants analyzed. This suggested that template DNArequired for amplification of the authentic DUF247 comprising M-locuslinked suppressor gene of gynoecium development (GDS) was lacking inthose three plants. To confirm this hypothesis genomic DNA has beensequenced using massive parallels sequencing for K1150-600-1,K323-600A6, and K1150.300-11 according to methods disclosed in EXAMPLE2. Mapping of reads, notably in the hemizygous M locus region, inspectedby using J-Browse indicate lack of female reads as in natural female(see FIG. 23). At regions flanking the hemizygous M-locus, loss ofheterozygosity is observed where the deletion overlaps with aheterozygous part of the chromosome. The determination of the correctborder of the deletions created is pending.

As female plants are also expected to naturally lack the M-locus linkedgynoecium development suppressor gene GDS which may occur spontaneouslyby an extremely small (but unneglectable) chance in the seed lots,plants were analyzed for their genetic purity. Template DNA obtainedfrom those individual plants was subjected to an microsatellite analysisusing 14 proprietary microsatellite markers (comparable to the design,use and discriminative power as outlined by Caruso et al., 2008; in factAO110 is their marker CV291890) and seven proprietary high resolutionmelting curve SNP markers which showed that 14 of the 16 plants capableof berry set certainly were authentic representatives of the hybridsthese belonged to. Two other plants showed a deviating microsatellitegenotype. One of those plants showed different alleles at all 14microsatellite loci and five SNP marker loci and because of thiscertainly was not an authentic member of the hybrid. Another plantshowed all the microsatellite alleles expected for the particular hybridto which it belonged with one notable exception, which was the lack ofthe paternal allele for the AO022 microsatellite marker, known to belinked to the M locus region. The typical single loss of the paternalallele of sex linked locus AO022 is expected to be indicative of theloss of a chromosomal segment that must have been lost as a result ofthe Cobalt 60 irradiation. This segment, at least for that particularplant, must span the region between the rising M-locus linked gynoeciumdevelopment suppressor (GDS) gene and microsatellite marker locus AO022.

An overview of the microsatellite analysis used to confirm theauthenticity of the mutants and their control hybrids is shown in FIG.21.

All plants that lacked the GDS gene fragments were further subjected tomarkers targeting genome scaffolds that were known to be positionedgenetically close or positioned in the M-locus region)

These primer pairs were:

CK63/64, CM45/46, CN96/97, CM98/99, CQ31/32, CT13/14, CE40/41 andCE64/CE66 (see Table 3). FIG. 17 shows an overview of the scaffolds (orscaffold parts) that could be mapped in the M-locus region. Depending onwhether the markers were informative it is indicated which extra part ofthe chromosomal segment, thus apart from the Gynoecium Developmentsuppressor gene that was already found to be lacking, is further missingin the irradiated plants capable of producing berries.

It appeared that three plants, for which a mutation event enabled themto produce berries, lack a chromosome segment on which both the GDS genedevelopment and the defective in tapetum development and function gene(TDF1) are located. As pointed out before: of two of these three plantsthe flowers were inspected and were proven to be of the female type thuswhich have flowers that have a fully developed gynoecium but furtherlack anthers. This provides evidence that a male plant can be convertedinto a female plant by ablation of both its GDS gene and the malestimulator or asparagus defective in tapetum development gene (AS-TDF1).The skilled person will appreciate that the opposite effect, whichcomprises the introduction of both these genes into a female plant willlikely result into a male plant. The skilled person will also appreciatethat by only introducing the defective in Tapetum Development andFunction gene (TDF1), thus not also including the DUF247 domaincomprising M-locus linked suppressor gene of gynoecium development, intoa female plant will change this female plant into a hermaphrodite plant.

Another independent, strong line of evidence that supports the TDF1 geneas being the male stimulator is an analysis of gene expression in allgenes displaying sex linkage. A ˜3.2 million SNP genetic map constructedusing 72 individuals of a doubled haploid mapping population delimited aregion of suppressed recombination on the Y that included 370 annotatedgene models. By calculating normalized gene expression values for all370 genes in this region of suppressed recombination (the M-locusregion), we first identified 11 genes that had expression values <1 FPKMin at least 3 of the 4 XX female libraries, a reasonable cutoff todetermine a gene as being non-expressed. Of these 11, we identify thegamma-irradiated DUF247 female suppression gene, and 10 putative malepromoting candidate genes. Candidates were first objectively pruned onthe basis of 1) expression in a female library, 2) presence of duplicategenes on an autosomal chromosome, 3) poor gene annotation (i.e.,mis-annotated retrotransposons), 4) gene expression and knockoutphenotypes in model systems. From the Harkess et al. (2015) study, onlyfour of the male and supermales libraries (89 male, 9 male, 89supermales, 103 male) were enriched with male reproductive geneexpression, likely a consequence of variation in reproductivedevelopment between breeding lines. These four libraries show consistentupregulation of three of the 10 putative candidates, Lipid TransferProtein DIR1, Tapetum Dysfunction 1 TDF1, and an Exopolygalacturonaseprotein. An LTP1 gene was found to have recombined in a breedingpopulation (CN94/CN95-11RM; primer see Table 3) Exopolygalacturonaseshave only been loosely related to anther activity, and are members of amulti-gene family in Asparagus, allowing for the possibility ofmis-aligned RNAseq reads due to high similarity between gene copies. TheTDF1 gene, on the other hand, is single copy in the Asparagus genome andonly present in this region of suppressed recombination on the Y.

The fact that AsOf TDF1-like is restricted to Male Asparagusofficinalis, thus is absent in Female Asparagus officinalis, is a singlecopy Gene Model, is in close vicinity of the Female suppressor genereferred to as DUF247 from AsOf_V2.0_scaffold905, is genetically flankedby several DNA-markers (such as CE64/CE66-HRM; Table 3) and is expressedat higher levels in Males and Supermales poses strong evidence that AsOfTDF1-like is the Male-promoting gene as predicted by the two-gene modelfor the origin of sex chromosomes (Charlesworth & Charlesworth, 1979).

The gene is referred to as AsOf TDF1.

The genetic pathway for tapetum development is generally conserved,given the similarity between Arabidopis thaliana and Oryza sativa (Caiet al., 2015, and references therein).

This is the case for both the crucial events of anther development, suchas sporophytic wall differentiation, tapetal specialization, meiosis andpollen maturation as well as for the crucial regulators of theseprocesses. In Arabidopsis and rice, transcription factors (TFs) that areessential for tapetum development and function have been identified. InArabidopsis these include the bHLH family members DYSFUNCTIONAL TAPETUM(DYT1) and ABORTED MICROSPORES (AMS), the R2R3 MYB TFs DEFECTIVE inTAPETAL DEVELOPMENT and FUNCTION (TDF1) and MS188/MYB0 and PHD-fingerprotein MALE STERILITY (MS1). Rice homologs for these TFs includeUNDEVELOPED TAPETUM (UDT1), TAPETUM DEGENERATION RETARDATION (TDR1),OsTDF1, OSMYB103/OsMYB80 and PERSISTENT TAPETUM CELL1 (PTC1). Theseregulators form a genetic pathwayDYT1/UDT1→TDF1/OsTDF1→AMS/TDR→MS188/OsMS188 MS1/PTC1 In which TDRinteracts with two other bHLH family members (bHLH142 and EAT1, see Caiet al., 2015). Both in Arabidopsis and rice, DYT1/UDT regulates the geneexpression for pollen wall development of all downstream genes,primarily via TDF1/OsTDF1. Two lines of evidence using gene expressiondata to support the AsOf TDF1 being the male promoter in Asparagus wereconducted: a forward genetic approach in which all genes displaying sexlinkage were analyzed and a reverse genetics approach in which theconserved genetic pathway mentioned was used to analyse the expressionof Asparagus homologs of the key regulators in Arabidopsis and rice.

The first approach (described in Harkess et al., 2015) a ˜3.2 millionSingle Nucleotide polymorphism (SNP) genetic map constructed using 72individuals of an Asparagus Officinalis DH mapping population (Limgroup,Horst, The Netherlands) delimited a region of suppressed recombinationon the Y-specific region of the sex chromosome that included 370annotated gene models. By calculating normalized gene expression valuesfor all 370 genes in this region of suppressed recombination, 11 geneswere not expressed in DH female lines; the DUF247 female suppressiongene (the SGD gene (identical to SEQ ID:NO 1 and SED NO 3), and 10putative male promoting candidate genes. Candidates were firstobjectively pruned on the basis of presence of duplicate genes on anautosomal chromosome, poor gene annotation (i.e., mis-annotatedretrotransposons) and gene expression and knockout phenotypes inArabidopsis and rice. Harkess et al. (2015) describe that only four ofthe male and supermales samples used in RNA-Seq experiments (89 male, 9male, 89 supermales, 103 male) show differential male reproductive geneexpression, likely a consequence of variation in reproductivedevelopment between breeding lines. The results show consistentupregulation of three of the 10 putative candidates that are Asparagushomologs of LIPID TRANSFER PROTEIN DIR1 LTP1), AsOf TDF1 (SEQ ID NO:4),and an Exopolygalacturonase protein. Exopolygalacturonases have onlybeen loosely related to anther activity, and are members of a multi-genefamily in Asparagus, allowing for the possibility of mis-aligned RNA-seqreads due to high similarity between gene copies. These results indicatethat AsOf TDF1 is involved in male-specific gene expression. The secondapproach used the Arabidopsis and Oryza sativa sequences of the keyregulators in the conserved genetic pathway for tapetum development toanalyse candidate homologous gene models in Asparagus Genome ScaffoldV2.0 (AGS V2.0) and annotation metadata. For this, tBLASTN was used withthe protein sequences of the key regulators as Query in BLAST databasesof AGS V2.0 and RNA-Seq Trinity de novo assemblies. The returnedsequences with significant similarity scores were inspected andevaluated by BLASTP in standard settings with the translations of thecandidates as Query in NCBI non-redundant protein databases ofArabidopsis thaliana and Oryza sativa.

For DYT1/UDT1 no significant tBLASTN hits were found in AGS V2.0 and onerelevant hit in the Trinity assemblies: comp64619_c4_seq3 of 847nt. SEQID: NO 10 SEQ ID NO:10 comp64619_c4_seq3 of 847nt

CTCTCTCTCTCTCTCTCTGCAATTTACAAGTACTTCTTCTCCGTTGCTTGTTAGCATTATTTGATAGCAATGCCTCGTTGGCCAAGAGACCAAGCCAAGGAATTTGATGTGATGAACTTCGCAGACTCAATGCTTGATGGCTGCTACGGCGATGGAGGAGGAGAAGGGGAGTTTCGGAAGGAGCAGTCCGCGGCTGCGGCAGAGAAGGGAGAGGAAAGGTACAAGTCAAAGAACCTCGCAGCAGAGAGGAGGAGGAGGAGCAAACTCAATCATCGACTCTTTACCCTCAGATCTTTGGTTCCTAACATTACTAAGATGAGCAAGGAGTCAACCCTCATTGATGCAATGGATTACATCCACAACCTCCAAACACAAATTAGTGACCTGAAGCTTGAGATTTCGAAGATTTGCGAAGAAGAGGACCGCACGAAGCAAGGGAGCACATCTAGTACAGAGAGCACAGCTCCTCCAGAGATGGCCCAATACCAGGGAAGGGTTGAGCTGAATCCTATGGGACAAAACAAATTCCATGTTAAGATTATGTGCAACAAGAGGCCTGGAGGGTTTATTAAACTGCTTGATGCCCTCTCCAGAAATGGACTAGAGATTACTGAAATCAGCTCCTTTGCTTTTTCAGGTTTTGATCAGATAGTTTTTTGCATTGAGGCAACGGGTGATAAGGAGATTCCCATTTCTGAGTTAAGAAAGCTTCTAATGGCGATAGTCGAAGTATCTGAGGAGAATAATAAATGATTAATTTTAAATCATGTTCAATTGGTATTTGTATGAATAGATTGATTTAGAGTTTGAACTTCAAAGTTTTCTGTGCTTTTATTTGCTTTAGTAA

When used as Query in BLASTP the top scoring sequences included the bHLHdomain in AMS/TDR1 and TF SCREAM2 in Arabidopsis. It was concluded thatDYT1/UDT1 has no significant homologous sequence in the used maledatabases.

For TDF1/OsTDF1, the homologous genomic sequence is described before andcan be found in SEQ ID NO: The female sequence is absent and theexpression is male-restricted upregulated (Harkess et al., 2015 andpersonal observations, Limgroup, Horst, The Netherlands).

For AMS/TDR1, one tBLASTN sequence was found in AGSV2.0:AsOf_V2.0scaffold2800 positions 121055 . . . 121735 with Identities73/227 (33%) and positives 98/227 (44%). The AMS/TDR1 predicted cDNA isprovided in SEQ ID NO:7

ATGAAGGTGTTGTCATATTCCAGCGTGGTTGAGGGTCTGAGGCCACTTGTGGGTGGCAATGGCTGGGACTACTGCATCCTGTGGAAATTGTCTCAAGATCAGAGGTTTTTGGAGTGGATGGGATGCTGTTGTAGCGGAACAGAGGCAAGCATTGCGAATGGTGGAGGGCTTTTCTCTGGTGATGAAACATTTCAGAAATCACCATGCAGGGATTTAATGCTGCAGCATCCAAGAACAAGGGCATGCGATGCTCTCTCAGAGTTTCCTTCTTCCATCCCCTTGGATTCCTCTTTAGGCATTTACGCACAAGTATTGATGTCGAACCAGCCAACTTGGCAAACACTTCATGATGCGGTTGGAGCAAAGACTAGGGTTCTTGTTCCTATTGCTGGTGGACTAGTTGAGCTACTAGTCTCGAAGCAAGTTGCTGAGAACCAACAGATGACAGACTTCATCATGTCACAATGCAACGGGAGCATCTACGACCATCCAACTGCGGGTAATTTCCTTGATGATCAGAGTTTCCAGTGGGAGGCATCCGCAGGTGGCCAATCACAACCCTACGCATCTCCGATGAACATCTTCGACCAGTTGCAGCTCGATGCGGCTGCAACAATGGACAGCACGGGGTACGGGCAGCAGGCAGGGCTGACGAGTGTGCATCAGCAAAAGGAATCTGCTCCAGCGGAGAAGGAATCGGTGAAACATGAGGGCGGCAGTGCGCGAGGAGATTCGGGGACGGAGGGGAGTGAGGATGATGAGGAGGGGAGGGCGGTAGGGAAGAACGGGAAGCGGCATCATGCAAAGAATCTTGTGGCGGAGAGGAAGAGGAGGAAGAAGCTTAATGATCGGCTCTACGCTCTCAGGGCCTTGGTTCCTAAAATCACAAAGATGGATAGAGCATCGATTCTTGGAGATGCGATAGAGTATGTGATGGAGTTACAGAAGCAGGTAAAAGATCTGCAGGACGAGCTCGAGAATGAATCAAATCCAGATGACACCGATTCAAAGCAAATCGAAAGCAACTATGACAATGTGGAAACAGGCAATCGAAATGGGATGATAAATTATAATCTCATGGAGCTTGAGGAGTCCCTTAACGCTACAAGTACGAGAAATGCTAAGACTGTTGATCAGTCGAACAATGAGGAGAAGGGGAATCAAATGGAGCCACAAGTGGAGGTGAAGCAGCTGGAAGCTAATGACTTCTACCTCAAGGTTTTTTGTGAGCATAAGGTTGGAGGATTTGCAAGGCTGATGGAGGCAATGAGCTCGCTTGGGCTGGAGGTGACCAATGCAAGTGTGACTACTCTTCAGTCTTTAGTACTGAATGTTTTCAGGGTGCAGAAGAGGGACAATGAAACGATGCAAGTCGATCAAGTCAGGGATTCATTGCTGGAGCTGACTCGAGGGCCAATCCGAGGGTGGCCGGAGCCTGGACACACTACAGAAAACCGCGGTGGAGATTGCCATCATGACAATGGTCTGCGGCCTACCGTGGAGATTTGGAGAATTTTGATTGTCGTGTTGTGCCAAGCTGGCAACGTTCCTTTGGGTTTTGGTTTGTTTGGAAAAATAATAGATTCGGGAAGTTTGCCGACTGTTGTGACGTATACGTTTCTTATTAAAGGGCTCCTAAAAGCTCGAATGTTGAGCGAAGCGATTGGTGTTTGGGATATTATGGTCATTGCCTCCGTTGCCGTCGACCGCCGCCTCGCCGCCCTCGACACGAAGCTATATTGA

Inspection of the alignment showed that the scores were the result ofalignment in the conserved bHLH family domain. This sequence wasdifferent from an AMS-related sequence described by Harkess et al.(Harkess et al., 2015). In this study the AMS candidate RNA wasmale-restricted downregulated as expected for AMS/TDR1-like sequences.Inspection of AsOf V2.0scaffold2800 female read coverage of referencefemale DH00/094 and four doubled haploid females showed no significantreduction in read coverage (results not shown) which indicates that theAMS gene is not lacking in females For MS188/OsMS188, one highlysignificant sequence was found using tBLASTN in AGS V2.0: AsOfV2,0_scaffold3320 positions 107598 . . . 106444 rev. The predicted cDNAof sequence MS188/OsMS188 is given in SEQ ID NO:8.

ATGGGAAGGATTCCTTGCTGTGAGAAGGATAATGTGAAGAGAGGACAGTGGACCCCCGAGGAGGACAACAAGCTCTCTTCCTACATCGCACAACACGGCACCCGAAACTGGCGTCTCATCCCCAAAAATGCCGGCCTTCAGAGATGTGGGAAGAGCTGCCGGCTACGATGGACCAACTACCTCCGCCCGGATCTCAAGCACGGCGTATTCTCAGACTCCGAAGAGCAGACCATCGTCAAGCTCCACTCCGTCGTCGGGAACAGGTGGTCGTTGATAGCAGGGCAACTGCCAGGGCGAACAGATAACGATGTGAAGAACCACTGGAACACGAAGCTGAAGAAGAAGCTGTTGGGCAAGGGTATCGACCCGGTGACCCACAAGCCCTTCTCCCATCTCATGGCCGAGATTGCTACCACGGTTCCCCCGCTGCAAGTAGCCCACCTCGCTGAAGCTGCCCTCGGCTGCTTCAAGGACGAAATGCTGCACCTCCTTACCAAGAAGCGGGCGGATTTCCCTGCAAACGGTACTGATGTCGGTGATGGCACGGGCTTCCCCTATGCAATGAGCCCCGTGGAGGACAAGGAAGAGACTGTTCAGAAGATCAAGCTAGGGCTCTCTCGAGCTATCATGCAGGAGCCTGGAACCGATAAGAGCTGGGGCTTAATGGAGAACGGAGAGCCATCAGATGGGCTTCCTGTTGTGTCAATGTGCGATGATGATTTGTATCGAACGATAGGGGATGAGTTCAGGTACGAGGGACCATCGTATGCGAATGGCGAGGGGTCAGCATGGAGCCAGAGCATGTGCACGGGTAGCACGTGCACTGGGGGCGGTGGAACACCAGACTGTCATGTATTGCACGAGAAACACAGTGACGACGAGGGGGTGGAGGCTGAAGGCAAGAGGAGGAAAATCGATGCTGGGCTTTTCGGCTCTGATGGTGTTTTATGGGATTTGTCTGATGACCTTATGATGAATCACATAG

Inspection shows near-full alignment of both protein sequences to theAsparagus homologous gene model and using BLASTP in non-redundantprotein databases at NCBI returned MS188 for Arabidopsis and OsMS188 ashighest scoring hits. In addition, the AsOf V2,0_scaffold3320 iswell-covered by female specific read mapping making it possible toanalyse gene expression in both males and females for this nonsex-linked gene model. The RNA-Seq data show a strict male-biasedexpression for the gene model i.e. the read mapping is absent in femaleexpression data. In the aforementioned RNA-Seq including in which thewhole genome gene expression in flower buds obtained from differentgenotypes of Asparagus and of particular developmental stages wasstudied. From these data it was concluded that AsparagusMS188/OsMS1.88-like gene model is exclusively expressed in malephenotypes restricted at the pre-meiotic stage. The gene model andspatiotemporal expression at the pre-meiotic stage corresponds well tothe MS188 and OsMS188 data (Gu et al., 2014, Cai et al., 2015).Therefore it was concluded that this gene model is the Asparagus homologof MS188/OsMS188. The gene is referred to as AsOf MS188.

For MS1/PTC1, the data are comparable to those of AsOf MYB188. Asignificant hit was returned using tBLASTN in AGS V2.0: AsOf V2.0scaffold2421 positions 133601 . . . 134341. The predicted cDNA sequenceMS1/PTC1 is given in SEQ ID NO:9:

ATGGAGAAGGTTCAATCTTGCTCTAGAAAGAGGAAAAGAGGAGAGAAGGTTTTCAGATTCGAGAGCTTCTGTGCACCTAGGCAACCAATACTTTTCAGTGGCTCGTTCCGAGACAACGTTAAGGCTCTTCTTGATTTCGGCCATCAAGAGGATGGAGTGCACGAAGGAATGCAGTTTTGGTCGTTTCGGCTCGAGCTTCATCAGTACCCTTCGACTTTCGTGAGGATGTTCGTTGCTGAGGAGGCTGTTGGGCTGTCGCAGAATCGCCAGTGCCTTTTTTGCCGATTCGCTGGTTGGGGGCACCACATGATCTCCAACAAGAGATTCCACTTCGTGCTGCCATTCAAAAAAACTAAATCAGAGGTCGAAAGCTTGAGCATAGAACTTGGTAGAAACAGACCAGGGATATCGTCAATGGGCTCGAAATTGATGGGTTCACAAGGAAAGCATCTAATGCATGGAATCATGCACTCTAATGGCTACGGACATCTCATTACTGTCAATGGCATTGAAGGAGGCTCTGATTTCATCTCTGGACATCAAATCATGGACTTGTGGGATAGGATTTGCACTGCTTTGCATGTGAGAAAAGTGAGTATAACAGATTCAGCAAAGAAGGGAAGCATGGAACTAAGGCTAATTCATGGACTAGTGTATGGTCAGCCCTGGTTCAGTCGCTGGGACTACAAACTAAGCCATGGAAGCTATGGCGTCACTCCCCAAATGTACCAAACCTCGCTCGAAGCCCTACGAACTCTCCCCTTATCAATCCTCCTCCCCAATTTCGCCTCTATCATTGCCAAGTACCAAACCCTAAGTGGGCTCAAGTTACAAACCATAGCCGACTTAACCTGCTTCATTACAGAGCTGAATCGTCGATTGCCCCCAAACACCCCTTCGACATTCGACTGTCGAGAAATCATCAGCGAGCCAACTTGTCGTTGGTCGATGAAACGAGTTGAGATGGCTGCTCAAGTCATAGTCGGGGCTCTAAAGAAGTCCAAATGTCGTTGGGTCACAAGACAAGAGGTCAGAGATGCCGCCAGAGCCTACATTGGTGACACAGGCCTACTAGACTACGTGCTCAAGTCTCTCGGCAACCACATTGTTGGAAACTATGTTGTTCGACGGATGGTCAACCCGATAACCAAAATACTTGAATACTGCTTGCAGGATGTATCTACTGTTTTCCCTAGCTTGGATCATTTCGGTTCACTTCGTTTTCATGTCACAAGGTCTCAGCTCAAGAAAGACATGATGTACCTCTACAATAACATATTTGGAGCACATAGCACATTGGCTGCCGATGGGGTTTTCAGGGCAATACTTATCGCTGCTCGGGTGATTCTCGACGCCAAACACCTTGTTAAGGATTACAAGGTGACAGGTGGCTCGTTACAAGACACCCAAATGAAGAACAATGATCAATGTTTAAAGGTAATGTGCACGATACGAATCATGAACAATCAAGAGAAGAAGGAACTGCCACCATATGAGATGTTCACCTTTCAGCTCAATGCAACAATTGGGGACCTGAAGAGAGAGACTGAAAAAAAGTTCAGGGAAATCTATTTGGGCCTGAAGAGCTTCACTGCAGAATCAGTGGCTGGTCTTAATGCTGAAGATACTGATTTCATTGTAGGAGTACTTGTTGAGCTTGGCAACAAAGTGATTGTTGAAGGAAGAGTAGTTAATAATGCTGATGAGATTTATGAGGGTGGAAAAGATGTGGATTGCCATTGCGGAGGGAAGGAGGAGGATGGAGAGGTGATGGTGTGCTGCGATATCTGTGGGATTTGGCAGCATGCAAGGTGTGCAGGGATTGAGGACGAAGAAGAGGATGTTCCTAGGGTTTTTCTCTGTAACCTATGCGAGAACAATATTTCCGCATTGCCTCCAA TTCAATACTAG

Inspection shows near-full alignment of both protein sequences to theAsparagus homologous gene model and using BLASTP in non-redundantprotein databases at NCBI returned MS1 for Arabidopsis and PTC1(Os09g0449000) as highest scoring hits. In addition, the AsOfV2,0_scaffold2421 is well-covered by female specific read mapping makingit possible to analyse gene expression in both males and females forthis non sex-linked gene model. The RNA-Seq data show a strictmale-biased expression for all four exons of the gene model i.e. theread mapping is absent in female expression data. Some aspecific readmapping occurs both in males and females. In the aforementioned RNA-Seqincluding in which the whole genome gene expression in flower budsobtained from different, genotypes of Asparagus and of particulardevelopmental stages was studied. From these data it was concluded thatthe Asparagus MS1/TCP1-like gene model is exclusively expressed in malephenotypes restricted at the pre-meiotic stage. The gone model andspatiotemporal expression at the pre-meiotic stage corresponds well todie MS1 and PCT data (Gu et al., 2014, Cai et al., 2015). Therefore itwas concluded that this gene model is the Asparagus homolog of MS1/PCT1.The gene is referred to as AsOf MS1. Notably, the male-biased, RNA-seqread mapping of AsOf MS1 is absent in line 9M (Limgroup, Horst, TheNetherlands). This was due to the small amount of flower buds sampledsome particular stages. In conclusion the regulatory network reveals:DYT1/UDT (no reliable predictions)→TDF1/OsTDF1/AsOfTDF1→AMS/TDR1(?)→MS188/OsMS188/AsOf MS188→MS1/PTC1/AsOf MS1

The fact that AsOf TDF1-like is restricted to Male Asparagusofficinalis, thus is absent in Female Asparagus officinalis, is a singlecopy Gene Model, is in close vicinity of the Female suppressor genereferred to as of the Gynoecium Development Suppressor (GDS gene) or theDUF247 domain containing gene from AsOf_V2.0_scaffold905, is geneticallyflanked by several DNA-markers, is expressed at higher levels in Malesand Supermales and is part of a well-studied genetic pathway for tapetumdevelopment for which Asparagus homologs show the expectedspatio-temporal expression patterns, poses strong evidence that AsOfTDF1-like is the male-promoting gene as predicted by the two-gene modelfor the origin of sex chromosomes (Charlesworth & Charlesworth, 1979).In addition, one can safely conclude that complementing a femaleasparagus plants with AsOf TDF1 will restore a functional androeciumdevelopment.

Cai et al (2015) have demonstrated the expression of OsTDF1 inArabidopsis tdf1 mutant restores its fertility, suggesting that thishomolog can fulfill the normal function of TDF1 in Arabidopsis. The riceOsTDF1 gene and the Arabidopsis TDF1 gene have been shown to be quitedifferent but conserved in the R2R3 MYB motif. This knowledge combinedwith the knowledge disclosed in the present document indicates that afemale asparagus plants with complemented with a homolog or ortholog ofAsOf TDF1 may also restore a functional androecium development.

TABLE 61 Result of BioNano Genomics contig assembly of Asparagusofficinalis genotype DH00/086 (Male) and AGS V2.0 Scaffolds using theIrysview Software ® package. Based on genetic marker information, 16 AGSV2.0 scaffolds were selected (sex-linked) as Query and yielded 8different BioNano contigs (7, 22, 28, 55, 438, 833, 1030, and 1138) orno contig (0). The table shows that 7 sex-linked scaffolds matched toBNG V1.0 contig 28 and 4 scaffolds not detected by genetic markerscreening (new) matched to contig 28 as well. Based on matchinginformation it was concluded that at least 7 M-locus scaffolds werechimeric assemblies. AGS V2.0 scaffold BNG V1.0 contig Genetic markerMatch Query_id Anchor_id information information 206 28 sex linkedchimeric 422 1030 sex linked 436 28 new chimeric 905 28 sex linked 94528 sex linked chimeric 997 222/28 sex linked chimeric 1139 7 sex linkedchimeric 1166 1138 sex linked chimeric 1194 28 sex linked 1204 28 sexlinked chimeric 1279 833 sex linked 1539 28 sex linked 1742 458 sexlinked 1761 0 sex linked 2312 28 sex linked chimeric 2510 28 new 3098 0sex linked 3294 28 new 3779 28 new 5266 0 sex linked

Example 7

Feminized Plants (Including Females) Created by Gamma Irradiation; theirFruit Set, their Flowers, their Proven Mutations

In the present EXAMPLE 7 more details are provided on the mutants plantobtained by gamma irradiation described in EXAMPLE 6

At the time of writing research was ongoing. The text below provides arecord of what is currently known. It should be noted that the plants atthe time of both the first and second evaluation have suffered from aProdiplosis longifila infection and that therefore the fruit set couldhave been higher, compared to what has been reported for those plants.The second evaluation took place in December 2015 in a warm period thatmay also negatively affect fruit set

For all mutants HRM analyses was performed on their DNA as described inEXAMPLE 6 which, apart from the male-to female transgenders, showed onlya melting curve difference for K1150_300_11 that indeed had a mutation(see below). To be certain that some mutations had not been missed (suchas A→T type 4 SNPs) by HRM, the gene region was sequenced for allmutations but K1150-600-2 (sent for massive parallel sequencing) usingprimers CN86/CN87, CN88/CN89, CP41/CN60, CN59/CN70, CN67/CN82, CN69/CN81(Table 3). Only one mutant showed a SNP in a sequence obtained byCN86/CN87 outside the translated region and the region targeted by theHRM marker. This illustrates that extending sequencing outside thetranslated region may allow the detection of more mutants. However, ashas been noted before a region upstream the gene, for which PAB BIOreads showed AT rich repetitive DNA flanked by GC rich island (resultsnot shown). A region comprising repeats may contain cis-regulatoryelements such as have been shown for the Arahidopsis Fwa gene (Soppe etal 2000). The authenticity of all mutants have at least been proven bymarkers AO008, AO022, AO058, AO069, AO097, AO110, AO145 and showed noimpurities apart from K323-_600A3. This number of loci is sufficient tocall any impurity (unpublished results). However, especially for the(female) mutants that were subjected to costly genome sequencing, moremarkers have been applied such as shown in FIG. 21.

K1150-600-1 is a female also described in EXAMPLE 6, it has shown adeletion comprising the GDS and AS-TDF1 gene. A first inspection itsfemale flowering was recorded but poorly photographed. Several weekslater, the plant again produced female flowers of which one has beenphotographed which is shown in Figure. 22, Berries were found on threestems, two bore 5 and one bore 4 berries, nine ripe berries provided 11viable seeds. Four months later, after cutting of the fern, the plantwas found to have produced 152 new berries that are currently ripening

K1150.600-2, produced 5 stems (having 4, 2, 14, 57, and 4 berries each)of which four were ripen that provided five viable seeds several weekslater a flower was photographed FIG. 22. The flower showed a style andstigma development that was not exceptional for this hybrid. Recently,K1150-600-2 produced 20 new berries that are ripening. Genomeresequencing suggest a small candidate deletion staring at position 1449to 2023 which, because of PCR failure using primers CN88/CN89, CN86/CN87and CN62/CN68 (Table 3), provided no conclusive evidence to date of suchdeletion. Sequencing the GDS region of this mutant is pending.

K323-600A-3, had no young flowers at the first time of evaluation andproduced four stems that had (21, Ca 100, 1 and 11 berries respectively.This mutant was later classified as false because it appeared a seedcontamination (FIG. 21).

K323 600A-4 has finished flowering and then was found to have producedthree stems with 2, 1, and 4 ripened berries respectively that provided8 viable seeds. No new berries have been obtained for K323 600A-4 in newshoot flush.

K1129.300-5 had one stalk producing two ripe berries from which twoviable seeds were obtained. A flower of the plant was obtained in a newflush (FIG. 22). The image showed a very well developed tri-lobularstigma, which was not observed on a reference flower of the hybrid.Recently, this plant was reported to have produced 26 new berries.

K1129-300-7 had produced one stem that comprised 3 ripe berries fromwhich 4 viable seeds have been obtained, its photograph shows a stylewith some stigma development (but likely less compared to K1129-300-5).Recent inspection of the plants new shoots revealed no new fruit set

K1129-300.8 was found to have produced one ripe berry and in a nextflush providing a single viable seed. The flower of this plant is shownin FIG. 22. It was noted that this flower also has a very well developedstigma. Sanger sequencing K1129.300-8 using primer pairs CN86/CN87revealed an adenine to thymine change identical to nucleotide position1160 of SEQ ID NO:3. This adenine to thymine change is separated by 665nucleotides from the adenine of the first predicted start codon of theGDS gene. This conclusion an adenine to thymine is inferred from acomparison sequence information obtained for the K1129 reference hybrid,and of other plants such as K1036 (the genotype of EXAMPLE 6) breedingline 9M, and hybrid K1150 reference genome doubled haploid DH00/086. Thelikelihood of detecting such a mutation by chance alone in this regionmust be extremely small and therefore it is anticipated that such amutation may have enabled K1129.300-8 to produce at least one berry.Further investigation is pending. Thus far, no new berries have beenobtained in the second flush of stems.

K1129-300-9 produced one ripe berry comprising one viable seed. Aphotograph taken revealed no marked style development (FIG. 22) and itso far has not been reported to produce new berries.

K1150-300.10 had a single stem on which three ripe berries were foundfrom which two viable seeds have been obtained. It showed a relativelylarge fruit. The plant so far was not reported to have any newlyproduced berries.

K1129-300.11 had three stems on which (1, 2, and 3) berries were foundfor which only one viable seed was obtained. A picture was taken of aflower from the second shoot flush (FIG. 22) which showed anexceptionally long style, nearly topping its anthers. To date, newshoots have not provided new berries. High resolution melting analysisusing primer pair CP41/CN72, produced an off-type melting curve forplant K1150_300_11 compared to other individuals belonging to cultivarK1150, Sanger Sequencing using the primer pairs CN69/CN81 (Table 3,EXAMPLE 1) revealed an adenine to a guanine change comparable to thepositon of 1193 of 1160 of SEQ ID NO:1. which leads to a asparagine (N)to serine (S) amino acid change. This SNP was absent in a sequenceobtained for the K1150_300 reference hybrid and many reference sequencessuch as DH00/086, hybrid K323 and 88M, 5375, 9M etc. and is consideredunique. Because this mutant, capable of producing berries, has the aminoacid changed in of gynoecium development suppressor this differentiatesit from the original K1150 which is not capable of producing berries.Accordingly, it is concluded that this particular mutation provides afeminized plant.

K1150.300-12 had two stems comprising 174 and 6 berries from which >200viable seeds were collected. The plants has finished flowering at thetime of inspection and after cutting the fern to obtain new shoots, theplant has not recovered. Further investigation will take place on twelveseedling currently growing in the greenhouse obtained from theseberries. Fortunately, tissue was taken for DNA isolation prior tocutting the fern and, as disclosed in EXAMPLE 6, a deletion comprisingthe GDS and the male stimulator gene was proven to exist. The lack ofnew flowers so far has hampered confirmation of its expected femalephenotype. Future research aimed at obtaining new flowers from thepedigree of this plant may further confirm the association between thedeletion and a female flower phenotype are pending.

K1150-300-13 had a stem on which three ripe berries were found fromwhich 11 viable seeds have been obtained. An image of one of its flowers(FIG. 22) showed a very long style. On recently formed new shoots 18 newberries have been reported

K1150-300-14 produced two stems on which 3 and 4 ripe berries were foundthat produced 6 viable seeds. A flower (from which part of the ovary wascut) is shown (FIG. 22). No berries, thus far, have been obtained fromnew shoots. No flowers to be photographed have been obtained.

K1150.300-15 had a stem on which a single ripen berry was found that didnot have (registered) viable seed.

K1150.300-16 had a stem on which a single ripen berry was foundcomprising two viable seeds. Is was recently found to have producedthree new berries on a the second flush of stems.

Recently, more flower have been collected for reference plants of hybridK1129. It was noted that those plants have not developed any style orvery small.

This suggest that the style development as has been shown for K1129.5and K1129-8 is quite exceptional

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The invention claimed is:
 1. An Asparagus plant in which functionalexpression of a DUF247 domain-containing protein with SEQ ID NO:2,encoded by a dominant Gynoecium Development Suppressor (GDS) gene havingSEQ ID NO:3, or having at least 95% sequence identity to SEQ ID NO:3 isreduced, wherein the functional expression is reduced by disruption ofthe GDS gene having SEQ ID NO:3 or having at least 95% sequence identitythereto.
 2. The Asparagus plant of claim 1, wherein said plant is theresult of a treatment with gamma irradiation.
 3. The Asparagus plant ofclaim 1, wherein the GDS gene is disrupted by deletion of the codinginformation after a nucleotide corresponding to nucleotide 567 of SEQ IDNO:1.
 4. The Asparagus plant of claim 1, wherein the GDS gene isdisrupted by a deletion of a nucleotide corresponding to thymine atposition 527 of SEQ ID NO:1.
 5. The Asparagus plant of claim 1, whereinthe GDS gene is disrupted by an adenine to guanine mutation at aposition that corresponds to position 1193 in SEQ ID NO:1, resulting inan exchange of an asparagine (N) to serine (S) at a positioncorresponding to position 398 of SEQ ID NO:2.
 6. The Asparagus plant ofclaim 1, wherein the GDS gene is disrupted by an adenine to thyminechange at a position corresponding to position 1160 of SEQ ID NO:3,which is 665 nucleotides upstream the first start codon of the GDS gene.7. The Asparagus plant of claim 1, wherein the GDS gene is disrupted bydeletion of the entire GDS gene corresponding to SEQ ID NO:3.
 8. TheAsparagus plant of claim 1, which has a defective Tapetal Developmentand Function 1 (TDF1) gene having SEQ ID NO:6, or having at least 95%sequence identity to SEQ ID NO:6, or a deletion of the TDF gene.
 9. TheAsparagus plant of claim 1, which expresses an Asparagus officinalisTapetal Development and Function 1 (TDF1) protein as depicted in SEQ IDNO:5.
 10. The Asparagus plant of claim 1, which is an Asparagusofficinalis plane.